TCR and peptides

Abstract

To provide new Wilms tumour 1 protein (WT1) epitopes, particularly WT1 epitopes restricted to common HLA alleles, and new T-cell receptors (TCRs) that bind to WT1 peptides when presented by a major histocompatibility complex (MHC).SOLUTION: A novel WT1 epitope, and a TCR comprising a specific amino acid sequence in the CDR region responsible for binding specificity to WT1, are provided. Moreover, T-cells expressing TCRs, capable of specifically targeting and killing cells that overexpress the WT1 protein, are also provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a T cell receptor (TCR) that binds to a peptide derived from Wilms tumor 1 protein (WT1) when it is presented by the major histocompatibility complex. In this regard, the present invention relates to a complementarity determining region (CDR) that specifically recognizes the WT1 peptide. The present invention further relates to an immunogenic peptide derived from WT1. [Background technology]

[0002] T cell receptor (TCR) gene therapy is based on the genetic transfer of high avidity tumor-specific TCR genes into T lymphocytes, thus allowing them to specifically target desired tumor-associated antigens, resulting in less toxic and more specific and effective therapy. This approach has shown promise in clinical trials. One of the main barriers that limits the use of TCR gene therapy for the clinical treatment of cancer is the lack of tumor-specific T cells and corresponding TCRs. Thus, the low availability of tumor-specific TCRs remains an unsolved problem that limits the widespread use of TCR-based immunotherapy approaches.

[0003] The majority of tumor-associated antigens (TAAs) are self-antigens, and therefore T cells specific for such molecules are destroyed or anergized due to central and peripheral tolerance. Nevertheless, natural tumor-specific T cells have been observed in healthy donors and patients, especially in patients with hematological malignancies, after allogeneic hematopoietic stem cell transplantation (allo-HSCT), where the frequency of tumor-specific lymphocytes correlated with disease amelioration (Kapp, M. et al. Bone Marrow Transplantation 43,399-410 (2009); and Tyler, EM et al. Blood 121,308-317 (2013)).

[0004] The selection of tumor antigens to be targeted by immunotherapeutic approaches remains a matter of debate. An ideal TAA would be highly expressed on tumor cells but minimally expressed in healthy tissues.

[0005] Wilms' tumor 1 (WT1) is an intracellular protein that encodes a zinc finger transcription factor that plays an important role in cell growth and differentiation (Yang, L. et al. Leukemia 21, 868-876 (2007)). WT1 is widely expressed on various hematological and solid tumors, but shows limited expression on various healthy tissues (e.g. gonads, uterus, kidney, mesothelium, progenitor cells in various tissues). Recent evidence suggests a role for WT1 in leukemogenesis and tumorigenesis.

[0006] Several ongoing clinical trials rely on the generation of cytotoxic T lymphocyte (CTL) responses following vaccination with WT1 peptides. However, despite the recognition that WT1 may be useful for immunotherapy, only a small number of WT1 epitopes that are restricted to a limited number of HLA alleles are currently used for vaccination purposes (Di Stasi, A. et al. Front. Immunol. (2015)). One such epitope is HLA-A * The WT1 126-134 epitope (RMFPNAPYL; SEQ ID NO: 255) is presented by the MHC encoded by the 0201 allele (i.e., the epitope is HLA-A * 0201 is restrictive).

[0007] HLA-A * The major histocompatibility complex (MHC) with the 0201 haplotype is expressed in the majority (60%) of the Caucasian population, and therefore HLA-A * 0201-restricted epitopes and corresponding TCRs are of interest. * TCRs that target 0201-restricted WT1 epitopes are particularly advantageous because immunotherapy using such TCRs may be broadly applicable.

[0008] The WT1 126-134 epitope has been widely studied in several studies, either alone or in combination with additional tumor antigens. However, recent reports have highlighted major concerns regarding the processing of this particular epitope, which may impair its application for immunotherapy. Notably, the WT1 126-134 epitope is more efficiently processed by the immune proteasome than the standard proteasome (Jaigirdar, A. et al. J Immunother. 39(3):105-16 (2016)), thereby suppressing the expression of WT1 in many HLA-A antigens that endogenously express WT1. * 0201 Poor recognition of tumor cell lines or primary leukemia cells.

[0009] Therefore, novel WT1 epitopes, especially those related to prevalent HLA haplotypes (e.g., HLA-A * There remains a need for WT1 epitopes presented by MHC having the .

[0010] One naturally processed HLA-A has been identified * The 0201-restricted epitope is WT1 37-45, which has the amino acid sequence VLDFAPPGA (SEQ ID NO: 157, see, e.g., Smithgall et al. 2001; Blood 98 (11 Part 1): 121a). However, little has been reported about the TCR amino acid sequence, especially the CDR sequence, specific for this peptide sequence (Schmitt, TM et al. (2017) Nat Biotechnol 35: 1188-1195).

[0011] Therefore, there remains a need for new WT1 epitopes, particularly WT1 epitopes restricted by common HLA alleles, and new TCRs capable of binding to WT1 epitopes. Summary of the Invention

[0012] The present inventors have identified a novel TCR that binds to WT1 peptide when it is presented by MHC. Furthermore, the present inventors have determined the amino acid sequence of the TCR, including the amino acid sequence of the CDR region responsible for the binding specificity to WT1. Furthermore, the present inventors have demonstrated that T cells expressing the TCR of the present invention specifically target and kill cells overexpressing WT1 protein. Furthermore, the TCR of the present invention binds to HLA-A * 0201 and HLA-B * 3501 or HLA-B * It has been shown that 3502 and other antigens are restricted to MHC encoded by HLA class 1 and 2 alleles common in Caucasian populations.

[0013] Thus, in a first aspect, the present invention provides a T cell receptor (TCR) that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by a major histocompatibility complex (MHC), comprising: (i) a CDR3α comprising the amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3), or a variant thereof having up to three amino acid substitutions, additions or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO:8), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (ii) a CDR3α comprising the amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO:41), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (iii) a CDR3α comprising the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (iv) a CDR3α comprising the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO:30), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (v) a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (vi) a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO:30), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (vii) a CDR3α comprising the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO:57), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (viii) a CDR3α comprising the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASTQTPYEQYF (SEQ ID NO:63), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (ix) a CDR3α comprising the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSTVGGEDYGYTF (SEQ ID NO:69), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (x) a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO:57), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xi) a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASTQTPYEQYF (SEQ ID NO:63), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xii) a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSTVGGEDYGYTF (SEQ ID NO:69), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xiii) a CDR3α comprising the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO: 80), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xiv) a CDR3α comprising the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO:86), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xv) a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSDTRAREQFF (SEQ ID NO:97), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xvi) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xvii) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xviii) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xix) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xx) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxi) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxii) a CDR3α comprising the amino acid sequence of CAVTVGNKLVF (SEQ ID NO: 175), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRGWREQFF (SEQ ID NO: 180), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxiii) a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSWGYQETQYF (SEQ ID NO: 196), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxiv) a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxv) a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxvi) a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSWGYQETQYF (SEQ ID NO: 196), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxvii) a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxviii) a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxix) a CDR3α comprising the amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxx) a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxi) a CDR3α comprising the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSDTRAREQFF (SEQ ID NO:97), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxii) a CDR3α comprising the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxiii) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxiv) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxv) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO:282), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxvi) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO:288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxvii) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO:288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxviii) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO:288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxix) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO:288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxx) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO:282), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxxi) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions, or deletions; or (xxxxii) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO:282), or a variant thereof having up to three amino acid substitutions, additions, or deletions; Provides TCR.

[0014] In one embodiment the invention provides a TCR of the invention comprising the following CDR sequences: (i) CDR1α - KALYS (SEQ ID NO: 1), CDR2α - LLKGGEQ (SEQ ID NO: 2), CDR3α - CGTAWINDYKLSF (SEQ ID NO: 3), CDR1β - SGHDY (SEQ ID NO: 6), CDR2β - FNNNVP (SEQ ID NO: 7), and CDR3β - CASRKTGGYSNQPQHF (SEQ ID NO: 8), or each of these variants having up to three amino acid substitutions, additions or deletions; (ii) CDR1α - NSASQS (SEQ ID NO: 34), CDR2α - VYSSGN (SEQ ID NO: 35), CDR3α - CVVNLLSNQGGKLIF (SEQ ID NO: 36), CDR1β - LGHNA (SEQ ID NO: 39), CDR2β - YSLEER (SEQ ID NO: 40), and CDR3β - CASSQDYLVSNEKLFF (SEQ ID NO: 41), or each of these variants having up to three amino acid substitutions, additions or deletions; (iii) CDR1α -SSVPPY (SEQ ID NO: 12), CDR2α - YTSAATLV (SEQ ID NO: 13), CDR3α - CAVRLSGSARQLTF (SEQ ID NO: 14), CDR1β - SGHAT (SEQ ID NO: 22), CDR2β - FQNNGV (SEQ ID NO: 23), and CDR3β - CASSLLGDEQYF (SEQ ID NO: 24), or each of these variants having up to three amino acid substitutions, additions or deletions; (iv) CDR1α -SSVPPY (SEQ ID NO: 12), CDR2α - YTSAATLV (SEQ ID NO: 13), CDR3α - CAVRLSGSARQLTF (SEQ ID NO: 14), CDR1β - SGHTA (SEQ ID NO: 28), CDR2β - FQGNSA (SEQ ID NO: 29), and CDR3β - CASSLVALQGAGEQYF (SEQ ID NO: 30), or each of these variants having up to three amino acid substitutions, additions or deletions; (v) CDR1α - TSESDYY (SEQ ID NO: 17), CDR2α - QEAYKQQN (SEQ ID NO: 18), CDR3α - CAYRSLKYGNKLVF (SEQ ID NO: 19), CDR1β - SGHAT (SEQ ID NO: 22), CDR2β - FQNNGV (SEQ ID NO: 23), and CDR3β - CASSLLGDEQYF (SEQ ID NO: 24), or each of these variants having up to three amino acid substitutions, additions or deletions; (vi) CDR1α - TSESDYY (SEQ ID NO: 17), CDR2α - QEAYKQQN (SEQ ID NO: 18), CDR3α - CAYRSLKYGNKLVF (SEQ ID NO: 19), CDR1β - SGHTA (SEQ ID NO: 28), CDR2β - FQGNSA (SEQ ID NO: 29), and CDR3β - CASSLVALQGAGEQYF (SEQ ID NO: 30), or each of these variants having up to three amino acid substitutions, additions or deletions; (vii) CDR1α - TSINN (SEQ ID NO: 45), CDR2α - IRSNERE (SEQ ID NO: 46), CDR3α - CATDAYSGNTPLVF (SEQ ID NO: 47), CDR1β - MNHNS (SEQ ID NO:55), CDR2β - SASEGT (SEQ ID NO:56), and CDR3β - CASRAAGLDTEAFF (SEQ ID NO:57), or each of these variants having up to three amino acid substitutions, additions or deletions; (viii) CDR1α - TSINN (SEQ ID NO: 45), CDR2α - IRSNERE (SEQ ID NO: 46), CDR3α - CATDAYSGNTPLVF (SEQ ID NO: 47), CDR1β - MNHNY (SEQ ID NO: 61), CDR2β - SVGAGI (SEQ ID NO: 62), and CDR3β - CASTQTPYEQYF (SEQ ID NO: 63), or each of these variants having up to three amino acid substitutions, additions or deletions; (ix) CDR1α - TSINN (SEQ ID NO: 45), CDR2α - IRSNERE (SEQ ID NO: 46), CDR3α - CATDAYSGNTPLVF (SEQ ID NO: 47), CDR1β - SGHNS (SEQ ID NO: 67), CDR2β - FNNNVP (SEQ ID NO: 68), and CDR3β - CASSTVGGEDYGYTF (SEQ ID NO: 69), or each of these variants having up to three amino acid substitutions, additions or deletions; (x) CDR1α - DSAIYN (SEQ ID NO: 50), CDR2α - IQSSQRE (SEQ ID NO:51), CDR3α - CAVRAEIYNQGGKLIF (SEQ ID NO:52), CDR1β - MNHNS (SEQ ID NO:55), CDR2β - SASEGT (SEQ ID NO:56), and CDR3β - CASRAAGLDTEAFF (SEQ ID NO:57), or each of these variants having up to three amino acid substitutions, additions or deletions; (xi) CDR1α - DSAIYN (SEQ ID NO: 50), CDR2α - IQSSQRE (SEQ ID NO:51), CDR3α - CAVRAEIYNQGGKLIF (SEQ ID NO:52), CDR1β - MNHNY (SEQ ID NO: 61), CDR2β - SVGAGI (SEQ ID NO: 62), and CDR3β - CASTQTPYEQYF (SEQ ID NO: 63), or each of these variants having up to three amino acid substitutions, additions or deletions; (xii) CDR1α - DSAIYN (SEQ ID NO: 50), CDR2α - IQSSQRE (SEQ ID NO:51), CDR3α - CAVRAEIYNQGGKLIF (SEQ ID NO:52), CDR1β - SGHNS (SEQ ID NO: 67), CDR2β - FNNNVP (SEQ ID NO: 68), and CDR3β - CASSTVGGEDYGYTF (SEQ ID NO: 69), or each of these variants having up to three amino acid substitutions, additions or deletions; (xiii) CDR1α - DSASNY (SEQ ID NO: 73), CDR2α - IRSNVGE (SEQ ID NO: 74), CDR3α - CAASMAGAGSYQLTF (SEQ ID NO: 75), CDR1β - ENHRY (SEQ ID NO:78), CDR2β - SYGVKD (SEQ ID NO: 79), and CDR3β - CAISVGQGALYEQYF (SEQ ID NO: 80), or each of these variants having up to three amino acid substitutions, additions or deletions; (xiv) CDR1α - DSASNY (SEQ ID NO: 73), CDR2α - IRSNVGE (SEQ ID NO: 74), CDR3α - CAASMAGAGSYQLTF (SEQ ID NO: 75), CDR1β - SGDLS (SEQ ID NO: 84), CDR2β - YYNGEE (SEQ ID NO: 85), and CDR3β - CASSVARDRRNYGYTF (SEQ ID NO: 86), or each of these variants having up to three amino acid substitutions, additions or deletions; (xv) CDR1α - NSMFDY (SEQ ID NO: 90), CDR2α - ISSIKDK (SEQ ID NO: 91), CDR3α - CAANNARLMF (SEQ ID NO: 92), CDR1β - SGHNS (SEQ ID NO: 95), CDR2β - FNNNVP (SEQ ID NO: 96), and CDR3β - CASSDTRAREQFF (SEQ ID NO: 97), or each of these variants having up to three amino acid substitutions, additions or deletions; (xvi) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xvii) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xviii) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xix) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xx) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxi) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxii) CDR1α - VGISA (SEQ ID NO: 173), CDR2α - LSSGK (SEQ ID NO: 174), CDR3α - CAVTVGNKLVF (SEQ ID NO: 175), CDR1β - MNHNS (SEQ ID NO: 178), CDR2β - SASEGT (SEQ ID NO: 179), and CDR3β - CASRGWREQFF (SEQ ID NO: 180), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxiii) CDR1α - VGISA (SEQ ID NO: 184), CDR2α - LSSGK (SEQ ID NO: 185), CDR3α - CAARSYNTDKLIF (SEQ ID NO: 186), CDR1β - SGHTS (SEQ ID NO: 194), CDR2β - YDEGEE (SEQ ID NO: 195), and CDR3β - CASSWGYQETQYF (SEQ ID NO: 196), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxiv) CDR1α - VGISA (SEQ ID NO: 184), CDR2α - LSSGK (SEQ ID NO: 185), CDR3α - CAARSYNTDKLIF (SEQ ID NO: 186), CDR1β - KGHSH (SEQ ID NO: 200), CDR2β - LQKENI (SEQ ID NO: 201), and CDR3β - CASSPTGGEYYGYTF (SEQ ID NO: 202), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxv) CDR1α - VGISA (SEQ ID NO: 184), CDR2α - LSSGK (SEQ ID NO: 185), CDR3α - CAARSYNTDKLIF (SEQ ID NO: 186), CDR1β - MNHEY (SEQ ID NO: 206), CDR2β - SVGAGI (SEQ ID NO: 207), and CDR3β - CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxvi) CDR1α - NSMFDY (SEQ ID NO: 189), CDR2α - ISSIKDK (SEQ ID NO: 190), CDR3α - CAASYNNARLMF (SEQ ID NO: 191), CDR1β - SGHTS (SEQ ID NO: 194), CDR2β - YDEGEE (SEQ ID NO: 195), and CDR3β - CASSWGYQETQYF (SEQ ID NO: 196), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxvii) CDR1α - NSMFDY (SEQ ID NO: 189), CDR2α - ISSIKDK (SEQ ID NO: 190), CDR3α - CAASYNNARLMF (SEQ ID NO: 191), CDR1β - KGHSH (SEQ ID NO: 200), CDR2β - LQKENI (SEQ ID NO: 201), and CDR3β - CASSPTGGEYYGYTF (SEQ ID NO: 202), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxviii) CDR1α - NSMFDY (SEQ ID NO: 189), CDR2α - ISSIKDK (SEQ ID NO: 190), CDR3α - CAASYNNARLMF (SEQ ID NO: 191), CDR1β - MNHEY (SEQ ID NO: 206), CDR2β - SVGAGI (SEQ ID NO: 207), and CDR3β - CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxix) CDR1α - NSMFDY (SEQ ID NO: 212), CDR2α - ISSIKDK (SEQ ID NO: 213), CDR3α - CAASGGRDDKIIF (SEQ ID NO: 214), CDR1β - MNHEY (SEQ ID NO: 217), CDR2β - SVGAGI (SEQ ID NO: 218), and CDR3β - CASSYSRTESTDTQYF (SEQ ID NO: 219), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxx) CDR1α - NSMFDY (SEQ ID NO: 90), CDR2α - ISSIKDK (SEQ ID NO: 91), CDR3α - CAANNARLMF (SEQ ID NO: 92), CDR1β - SGHRS (SEQ ID NO: 269), CDR2β - YFSETQ (SEQ ID NO: 270), and CDR3β - CASSPGQHGELFF (SEQ ID NO: 271), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxi) CDR1α - NSMFDY (SEQ ID NO: 264), CDR2α - ISSIKDK (SEQ ID NO: 265), CDR3α - CAASATGNQFYF (SEQ ID NO: 266), CDR1β - SGHNS (SEQ ID NO: 95), CDR2β - FNNNVP (SEQ ID NO: 96), and CDR3β - CASSDTRAREQFF (SEQ ID NO: 97), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxii) CDR1α - NSMFDY (SEQ ID NO: 264), CDR2α - ISSIKDK (SEQ ID NO: 265), CDR3α - CAASATGNQFYF (SEQ ID NO: 266), CDR1β - SGHRS (SEQ ID NO: 269), CDR2β - YFSETQ (SEQ ID NO: 270), and CDR3β - CASSPGQHGELFF (SEQ ID NO: 271), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxiii) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxiv) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxv) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxvi) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxvii) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxviii) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxix) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxx) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxxi) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions; or (xxxxii) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions.

[0015] In one aspect the invention provides a TCR of the invention comprising: (i) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (ii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (iii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (iv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (v) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (vi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (vii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (viii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (ix) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (x) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO:53, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO:64, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xiii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xiv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xvi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xvii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xviii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xix) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xx) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 176, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 181, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxiii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxiv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 203, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 209, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxvi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 203, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxviii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 209, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; or (xxix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 215, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 220, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxx) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 272, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 267, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 267, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 272, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxiii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxiv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO:278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO:283, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxvi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO:278, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO:289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxviii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxx) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxxi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; or (xxxxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto.

[0016] In one embodiment the invention provides a TCR of the invention comprising: (i) an α chain comprising the amino acid sequence of SEQ ID NO:5, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, and variants of SEQ ID NOs:10 and 11 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (ii) an α chain comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, and variants of SEQ ID NOs: 43 and 44 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (iii) an α chain comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, and variants of SEQ ID NOs: 26 and 27 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (iv) an α chain comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and variants of SEQ ID NOs: 32 and 33 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (v) an α chain comprising the amino acid sequence of SEQ ID NO:21, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, and variants of SEQ ID NOs:26 and 27 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (vi) an α chain comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and variants of SEQ ID NOs: 32 and 33 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (vii) an α chain comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, and variants of SEQ ID NOs: 59 and 60 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (viii) an α chain comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66, and variants of SEQ ID NOs: 65 and 66 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (ix) an α chain comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 72, and variants of SEQ ID NOs: 71 and 72 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (x) an α chain comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, and variants of SEQ ID NOs: 59 and 60 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xi) an α chain comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66, and variants of SEQ ID NOs: 65 and 66 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xii) an α chain comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 72, and variants of SEQ ID NOs: 71 and 72 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xiii) an α chain comprising the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, and variants of SEQ ID NOs: 82 and 83 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xiv) an α chain comprising the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 88, SEQ ID NO: 89, and variants of SEQ ID NOs: 88 and 89 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, and variants of SEQ ID NOs: 99 and 100 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xvi) an α chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xvii) an α chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xviii) an α chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xix) an α chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xx) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxi) an α chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxii) an α chain comprising the amino acid sequence of SEQ ID NO: 177, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 182, SEQ ID NO: 183, and variants of SEQ ID NOs: 182 and 183 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxiii) an α chain comprising the amino acid sequence of SEQ ID NO: 188, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 198, SEQ ID NO: 199, and variants of SEQ ID NOs: 198 and 199 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxiv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 204, SEQ ID NO: 205, and variants of SEQ ID NOs: 204 and 205 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, and variants of SEQ ID NOs: 210 and 211 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 198, SEQ ID NO: 199, and variants of SEQ ID NOs: 198 and 199 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 204, SEQ ID NO: 205, and variants of SEQ ID NOs: 204 and 205 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, and variants of SEQ ID NOs: 210 and 211 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; or (xxix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 216, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 221, SEQ ID NO: 222, and variants of SEQ ID NOs: 221 and 222 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxx) an alpha chain comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 273, SEQ ID NO: 274, and variants of SEQ ID NOs: 273 and 274 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 268, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, and variants of SEQ ID NOs: 99 and 100 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 268, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 273, SEQ ID NO: 274, and variants of SEQ ID NOs: 273 and 274 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxxiii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxxiv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxv) an alpha chain comprising the amino acid sequence of SEQ ID NO:279, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:284, SEQ ID NO:285, and variants of SEQ ID NOs:284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxxvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxxvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxxviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; (xxxix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxx) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto; (xxxxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; or (xxxxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto.

[0017] In one embodiment, the invention provides a TCR of the invention comprising an alpha chain comprising the amino acid sequence of SEQ ID NO: 257, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising the amino acid sequence of SEQ ID NO: 259, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto.

[0018] In one embodiment, the invention provides a TCR of the invention comprising an alpha chain comprising the amino acid sequence of SEQ ID NO: 261, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence identity thereto; and a beta chain comprising the amino acid sequence of SEQ ID NO: 263, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity thereto.

[0019] The TCR of the present invention can bind to a WT1 peptide comprising an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123), YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254), and their respective variants having up to three amino acid substitutions, additions or deletions.

[0020] In another aspect, the invention provides a T cell receptor (TCR) that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), wherein the WT1 peptide is selected from the group consisting of EPASQHTLRSG (SEQ ID NO:123), YESDNHTTPIL (SEQ ID NO:126), NHTTPILCGAQYRIH (SEQ ID NO:127), QCLSAFTVHFSGQFT (SEQ ID NO:118), EDPMGQQGSLGEQQY (SEQ ID NO:119), SQLECMTWNQMNLGA (SEQ ID NO:120), APVLDFAPPGA (SEQ ID NO:117), NQMNLGATLKG (SEQ ID NO:250), DPGGIWAKLGAAEAS (SEQ ID NO:251), NHTTPILCGAQYRIH (SEQ ID NO:252), KRHQRRHTGVKPFQC (SEQ ID NO:253), PSCQKKFARSDELVR (SEQ ID NO: 254), and respective variants thereof having up to three amino acid substitutions, additions or deletions.

[0021] In one embodiment, the TCR of the present invention binds to MHC I and / or MHC II peptide complexes.

[0022] In one embodiment, the TCRs of the invention are restricted to human leukocyte antigen (HLA) alleles. In one embodiment, the TCRs of the invention are restricted to HLA-A or HLA-B alleles. In one embodiment, the TCRs of the invention are restricted to HLA-A or HLA-B alleles. * 0201, HLA-A * 0101, HLA-A * 2402 and HLA-A * 0301, or HLA-B * 0702, HLA-B * 3501 and HLA-B * 3502.

[0023] In one embodiment, the TCR of the present invention is HLA-A * Arrested at 0201.

[0024] In one embodiment, the TCR of the present invention is HLA-B * Detained at 3502.

[0025] In one embodiment, the TCR of the present invention is HLA-B * Detained at 3501.

[0026] In one embodiment, the TCRs of the present invention are restricted to HLA-C alleles. * 07:01, HLA-C * 03:04, HLA-C * 04:01, HLA-C * 05:01, HLA-C * 06:02 and HLA-C * 07:02.

[0027] In one embodiment, the TCR of the invention comprises one or more mutations at the α chain / β chain interface such that when the α chain and β chain are expressed in a T cell, the frequency of mispairing between the chains and the endogenous TCR α and β chains is reduced.

[0028] In one embodiment, the TCR of the present invention comprises one or more mutations at the α chain / β chain interface such that when the α chain and β chain are expressed in a T cell, the expression levels of the TCR α chain and β chain are increased.

[0029] In one embodiment, the one or more mutations introduce a cysteine ​​residue into the constant region domain of each of the α and β chains, where the cysteine ​​residue is capable of forming a disulfide bond between the α and β chains.

[0030] The TCRs of the present invention may comprise murinized constant regions.

[0031] In one embodiment, the TCR of the present invention is a soluble TCR.

[0032] In another aspect, the present invention provides an isolated polynucleotide encoding the α chain of a T cell receptor (TCR) of the present invention, and / or the β chain of a TCR of the present invention.

[0033] In another aspect, the present invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:256 and / or the nucleotide sequence of SEQ ID NO:258, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0034] In another aspect, the present invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:260 and / or the nucleotide sequence of SEQ ID NO:262, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0035] In one embodiment, the isolated polynucleotide encodes an alpha chain linked to a beta chain, hi one embodiment, the isolated polynucleotide encodes one or more small interfering RNA (siRNA) sequences and / or one or more other factors capable of reducing or preventing expression of one or more endogenous TCR genes.

[0036] In another aspect, the invention provides a vector comprising a polynucleotide of the invention, hi one embodiment, the vector comprises a polynucleotide encoding one or more of the CD3 chains, CD8, a suicide gene, and / or a selectable marker.

[0037] In another aspect, the present invention provides a cell comprising a TCR of the invention, a polynucleotide of the invention, or a vector of the invention.

[0038] In one embodiment, the cells further comprise vectors encoding one or more of the CD3 chains, CD8, a suicide gene, and / or a selectable marker.

[0039] In one embodiment, the cell is a T cell, lymphocyte, or stem cell, such as a hematopoietic stem cell or an induced pluripotent stem cell (iPS). The T cell, lymphocyte, or stem cell may be selected from the group consisting of CD4 cells, CD8 cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, double negative T cells, naive T cells, memory stem T cells, central memory T cells, effector memory T cells, effector T cells, hematopoietic stem cells, and pluripotent stem cells.

[0040] In one embodiment, the cells are T cells isolated from a subject.

[0041] In one embodiment, preferably the endogenous gene encoding the TCR alpha chain and / or the endogenous gene encoding the TCR beta chain in the cell is disrupted such that the endogenous gene encoding the TCR alpha chain and / or the endogenous gene encoding the TCR beta chain is not expressed. In one embodiment, the endogenous gene encoding the TCR alpha chain and / or the endogenous gene encoding the TCR beta chain is disrupted by insertion of an expression cassette comprising a polynucleotide sequence encoding a TCR of the invention. In one embodiment, one or more endogenous genes encoding MHC in the cell are disrupted, preferably the cell is a non-allo-reactive universal T cell. In one embodiment, an endogenous gene involved in persistence, amplification, activity, resistance to exhaustion / senescence / inhibitory signals, homing capability, or other T cell function in the cell is disrupted, preferably the endogenous gene involved in persistence, amplification, activity, resistance to exhaustion / senescence / inhibitory signals, homing capability, or other T cell function is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160, and CTLA4. In one embodiment, the endogenous gene involved in persistence, amplification, activity, resistance to exhaustion / senescence / inhibitory signals, homing capability, or other T cell function is disrupted by integration of an expression cassette, the expression cassette comprising a polynucleotide sequence encoding a TCR of the invention.

[0042] In another aspect, the invention provides a method of preparing a cell, the method comprising the step of introducing a vector of the invention into a cell in vitro, ex vivo or in vivo, e.g. by transfection or transduction.

[0043] In another aspect, the invention provides a method of preparing a cell, the method comprising transducing the cell in vitro, ex vivo or in vivo with one or more vectors of the invention.

[0044] In one embodiment, the cells to be transduced with one or more vectors are selected from the group consisting of T cells, lymphocytes or stem cells, e.g., hematopoietic stem cells or induced pluripotent stem cells (iPS), and optionally the T cells, lymphocytes or stem cells may be selected from the group consisting of CD4 cells, CD8 cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, double negative T cells, naive T cells, memory stem T cells, central memory T cells, effector memory T cells, effector T cells, hematopoietic stem cells and pluripotent stem cells.

[0045] In one embodiment, the method comprises a T cell editing step, which comprises disrupting an endogenous gene, e.g. an endogenous gene encoding a TCR alpha chain and / or an endogenous gene encoding a TCR beta chain, using an artificial nuclease, preferably the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

[0046] In one embodiment, the method comprises a T cell editing step, which comprises disrupting the endogenous gene encoding the TCR α chain and / or the endogenous gene encoding the TCR β chain using an artificial nuclease, preferably the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

[0047] In one embodiment, the method comprises the step of targeted integration of an expression cassette into an endogenous gene encoding a TCR α chain gene and / or an endogenous gene encoding a TCR β chain gene that has been disrupted by an artificial nuclease, wherein the expression cassette comprises a polynucleotide encoding a TCR of the invention or a polynucleotide sequence of the invention.

[0048] In one embodiment, the method comprises disrupting one or more endogenous genes encoding MHC, and preferably the cells prepared by this method are non-allo-reactive universal T cells.

[0049] In one embodiment, the method comprises the step of disrupting one or more endogenous MHC genes, and preferably the cells prepared by this method are non-allo-reactive universal T cells.

[0050] In one embodiment, the method comprises disrupting one or more endogenous genes to alter persistence, amplification, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell function, preferably the method comprises targeted integration of an expression cassette into an endogenous gene involved in persistence, amplification, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell function that has been disrupted by an artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding a TCR of the invention, preferably the endogenous gene is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160 and CTLA4.

[0051] In another aspect, the invention provides a cell of the invention, or a cell prepared by a method of the invention, for use in adoptive cell transfer, preferably adoptive T cell transfer, optionally which may be allogeneic adoptive T cell transfer, universal non-allo-reactive T cell transfer, or autologous adoptive T cell transfer.

[0052] In another aspect, the present invention provides a TCR of the invention, an isolated polynucleotide of the invention, a vector of the invention, a cell of the invention, a cell prepared by the method of the invention, or a chimeric molecule of the invention for use in therapy.

[0053] In another aspect, the present invention provides a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell of the present invention, or a cell prepared by a method of the present invention, for use in the treatment and / or prevention of a disease associated with expression of WT1.

[0054] In another aspect, the invention provides genetically engineered (gene edited) T cells to alter persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capability, or other T cell function, wherein the T cells express a TCR alpha chain of the invention and / or a TCR beta chain of the invention.

[0055] In another aspect, the invention provides genetically engineered (gene-edited) T cells by a protocol comprising the targeted integration of an expression cassette into an endogenous gene disrupted by an artificial nuclease that is involved in persistence, amplification, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell function, wherein the expression cassette comprises a polynucleotide sequence encoding a TCR alpha chain of the invention and / or a TCR beta chain of the invention.

[0056] In another aspect, the present invention provides a method for treating and / or preventing a disease associated with expression of WT1, the method comprising the step of administering a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell of the present invention, a cell prepared by the method of the present invention, or a chimeric molecule of the present invention to a subject in need thereof.

[0057] The disease associated with the expression of WT1 may be a proliferation disorder. Preferably, the proliferation disorder may be selected from the group consisting of blood tumors, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma. The proliferation disorder may be selected from the group consisting of solid tumors, such as lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial carcinoma, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0058] In a preferred embodiment, the disease associated with expression of WT1 is acute myeloid leukemia (AML).

[0059] In another preferred embodiment, the disease associated with expression of WT1 is chronic myeloid leukemia (CML).

[0060] In another aspect, the present invention provides an isolated immunogenic WT1 peptide comprising an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO: 123), YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254), and variants thereof, each having up to three amino acid substitutions, additions, or deletions. [Brief description of the drawings]

[0061] [Figure 1-1]Plots showing the results of in vitro expansion of functional WT-1-specific T cells from peripheral blood of 10 healthy donors. Peripheral blood mononuclear cells of 10 healthy donors (HD) were stimulated with pooled overlapping WT1 15mer peptides for 26-30 h, enriched for CD137+ cells, and expanded for 9-19 days. The expanded T cells were restimulated with autologous antigen-presenting cells (APC) loaded with irrelevant peptide pools or WT1 peptide pools for 6 h. In addition, negative (unstimulated T cells) and positive (T cells cultured in the presence of PMA and ionomycin) controls were included in the experimental set-up (not shown). Dot plots show the results of intracellular staining for IFNγ production and CD107a exposure at the cell surface. After several restimulations with autologous APC loaded with WT1 peptide pools, T cell specificity was tested by intracellular staining as previously described. Results showed enrichment of WT1-specific T cells in the CD8 T cell compartment for HD1 (a), HD3 (c), HD4 (d), HD5 (e), HD6 (f), HD7 (g), and HD10 (j), and in the CD4 T cell compartment for HD2 (b), HD8 (h), and HD9 (i). WT1, Wilms tumor 1; PMA, phorbol 12-myristate 13-acetate; IFNγ, interferon-γ; S, stimulation. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 1-5] Continued from Figure 1-4. [Figure 1-6] Continued from Figure 1-5. [Figure 1-7] Continued from Figure 1-6. [Figure 1-8] Continued from Figure 1-7. [Figure 1-9] Continued from Figure 1-8. [Figure 1-10] Continued from Figure 1-9. [Figure 2-1]Grids and plots showing the identification of WT1 immunogenic peptides by the mapping grid strategy. Epitopes recognized by T cells primed in vitro by repeated stimulation with a pool of overlapping WT1 peptides were identified by intracellular staining. In particular, the percentage of specific T cells responding to the mapping grid of subpools of WT1 pentadecapeptide loaded on APCs was evaluated. In addition, negative (unstimulated T cells and T cells cocultured with APCs loaded with irrelevant peptide pools) and positive (T cells cultured in the presence of PMA and ionomycin) controls were included in the experimental setup (unstimulated T cells and PMA / ionomycin conditions are not shown). (a) Deconvolution grid showing the percentage of T cells expressing IFNγ and CD107a after coculture with APCs loaded with different subpools (denoted SP1-24). IFNγ and CD107a values ​​in bold indicate the subpools containing the WT1 epitopes recognized by T cells. Representative dot plots showing IFNγ and CD107a expression in association with co-culture of T cells with APCs loaded with responsive subpools are reported. Dominant responses were observed for subpools 4, 5, 16 in HD1 (b), HD3 (d), HD6 (g), HD7 (h), HD10 (k); subpools 6, 16, 17, 20, 23 in HD2 (c); subpools 4, 5, 6, 14, 18, 21 in HD4 (e); subpools 5, 11, 12, 21, 22 in HD5 (f); subpools 12, 14 in HD8 (i); and subpools 5, 13, 21 in HD9 (j). For HD7, we also observed increased IFNγ secretion and CD107a expression in response to subpools 7, 8, 20, albeit at a lower percentage compared to the responses observed with subpools 4, 5, 16. SP, subpool; WT1, Wilms tumor 1; APC, antigen-presenting cell; PMA, phorbol 12-myristate 13-acetate; IFNγ, interferon-γ. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 2-5] Continued from Figure 2-4. [Figure 2-6] Continued from Figure 2-5. [Diagram 2-7] Continued from Figure 2-6. [Figure 2-8] Continued from Figure 2-7. [Figure 2-9] Continued from Figure 2-8. [Figure 2-10] Continued from Figure 2-9. [Figure 2-11] Continued from Figure 2-10. [Figure 3-1]Epitope specificity of WT1-specific T cells generated by sensitization with pooled peptides. To verify WT1 immunogenic peptides, expanded T cells from each HD were co-cultured for 6 hours in the presence of APCs loaded with peptides identified after deconvolution of the mapping grid and at least one irrelevant peptide as a negative control. In addition, negative (unstimulated T cells) and positive (T cells cultured in the presence of PMA and ionomycin) controls were included in the experimental setup (not shown). Dot plots show the results of intracellular staining for IFNγ and / or surface CD107a for each HD. Enrichment of CD107a and / or IFNγ positive cells was observed, respectively, for HD1 (a) in T cells co-cultured with peptides 40 and 41, but not for peptides 42 and 43 (an irrelevant peptide); for HD2 (b) in T cells co-cultured with peptides 54, 77, 90, but not for peptides 42 and 138 (an irrelevant peptide); for HD3 (c) in T cells co-cultured with peptide VLDFAPPGA (SEQ ID NO: 157, VLD, which is a nonamer of the peptide represented by SEQ ID NO: 117 (referred to as "11mer" in FIG. 3c)), and peptides PVLDFAPPG (SEQ ID NO: 158, PVL, which is another nonamer of the peptide represented by SEQ ID NO: 117) and LDFAPPGAS (an immunogenic peptide previously described (Doubrovina, (E. et al., (2012), Blood, 120:1633-1646), low responses were observed for T cells co-cultured with peptide SEQ ID NO:159, a nonamer of the peptide represented by SEQ ID NO:116 (LDF); for HD4 (d, e), low responses were observed for T cells co-cultured with peptides 17, 18, 99, 100, but not for irrelevant peptides (15, 16, 63-66, 101, 102 and 132); for HD5 (f), low responses were observed for T cells co-cultured with peptide 101, but not for irrelevant peptides (63, 107, 108, 113, 119 and 120);For HD6(g), observed for T cells co-cultured with peptide VLDFAPPGA (SEQ ID NO: 157, VLD, which is a nonamer of the peptide represented by SEQ ID NO: 117 (referred to as "11mer" in FIG. 3c)) and peptide PVLDFAPPG (SEQ ID NO: 158, PVL, which is another nonamer of the peptide represented by SEQ ID NO: 117), but not for peptide LDFAPPGAS (SEQ ID NO: 159, LDF, which is a nonamer of the peptide represented by SEQ ID NO: 116, already described as an immunogenic peptide (Doubrovina, E. et al., (2012), Blood, 120:1633-1646); for HD9(h), observed for T cells co-cultured with peptides 101, 125, 137;For HD10(i), we observed T cells co-cultured with peptide VLDFAPPGA (SEQ ID NO: 157, VLD, which is a nonamer of the peptide represented by SEQ ID NO: 117 (referred to as "11mer" in FIG. 3c)) and none for irrelevant peptides. For HD7 and HD8, due to the reduced fitness of T cells, we could not perform functional tests to validate the peptides predicted by deconvolution of the mapping grid, i.e., peptides 40, 41, 91, 92 for HD7 and peptide 24 for HD8. To determine the HLA restriction of the WT1 epitopes identified for HD4, HD5 and HD10 T cells, donor DNA was sequenced to determine HLA typing. WT1-specific T cells were then co-cultured with different antigen-presenting EBV-BLCL cell lines, each one carrying the specific HLA allele of interest identified by sequencing of HD4, HD5 or HD10 DNA. EBV-BLCL cells were pulsed with peptide 17 for HD4, peptide 101 and peptide VLDFAPPGA (SEQ ID NO: 157) or an irrelevant control peptide for HD5. After 6 hours of co-culture, we observed substantial responses to WT1 by WT1-specific T cells co-cultured with EBV-BLCL cells expressing the HLA-B*3502 allele pulsed with peptide 17 for HD4 (j), EBV-BLCL cells expressing the HLA-B*3501 allele pulsed with peptide 101 for HD5 (k), and EBV-BLCL cells expressing the HLA-A*0201 allele pulsed with peptide VLDFAPPGA (SEQ ID NO: 157) for HD10 (l). (m) Table showing peptides recognized by T cells expanded from HD1-HD10. For HD3, HD6, and HD10, peptides 40 and 41 are shown, which overlap specific nonamers and induce immune responses. Wilms tumor 1; APC, antigen-presenting cell; PMA, 2; phorbol 12-myristate 13-acetate; IFNγ, interferon-gamma; S, stimulation. ; [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Diagram 3-4]Continued from Figure 3-3. [Figure 3-5] Continued from Figure 3-4. [Diagram 3-6] Continued from Figure 3-5. [Diagram 3-7] Continued from Figure 3-6. [Diagram 3-8] Continued from Figure 3-7. [Diagram 3-9] Continued from Figure 3-8. [Figure 3-10] Continued from Figure 3-9. [Figure 3-11] Continued from Figure 3-10. [Figure 3-12] Continued from Figure 3-11. [Figure 3-13] Continued from Figure 3-12. [Figure 4-1]Graphs and plots showing that HD1, HD3 and HD4 expanded T cells recognize naturally processed WT1 epitopes. (a) Graph depicting CD107 expression by CD8+ T cells expanded from HD1 after co-culture with T2 cells pulsed with a WT1 pool, K562 cells genetically modified to express HLA-A*0201 alleles and overexpress WT1 protein, or T2 cells pulsed with a non-specific control MelanA / MART1 pool as a negative control. (b) Graph depicting the results of an experiment to determine the ability of HD3 expanded T cells to target WT1 expressing cells. Results are expressed as a shedding index, which is calculated as the total number of target cells still present after co-culture with WT1 specific T cells divided by the total number of target cells alone. HD3 T cells were co-cultured with T2 cells pulsed with subpool 16 (SP16) containing immunogenic peptides to elicit an immune response, with MelanA / MART1 pool (MelanA) as a negative control, and with K562 cells that were either wild type (K562) or genetically modified to express the HLA-A*0201 allele and overexpress the WT1 protein (K562 A2+WT1+). (c) Plot depicting the results of an experiment to determine the ability of WT1-specific T cells from HD4 to eliminate target cells. HD4 T cells were co-cultured with primary CD33+ blasts collected from an HLA-B*3502 patient at a ratio of 10:1, or with leukemia cells from a patient without the HLA-B*3502 allele as a control. After 3 days of co-culture, results show near complete clearance of CD33+HLA-B*3502 blasts when seeded with WT1-specific T cells (CD3+ cells). E, effector; T, target. [Figure 4-2] Continued from Figure 4-1. [Diagram 5]Graph showing the results of Vβ profiling of WT1-specific T cells. After several rounds of stimulation with the WT1 pool, WT1-specific T cells arising from different HDs were stained with a Vβ immunoprofiling kit to determine the clonality of the population. In particular, the expression of variable (V) genes of the β chain was determined by FACS analysis. The results showed highly dominant expression of Vβ genes in HD1 (TRBV12-3;12-4), HD2 (TRBV11-2), HD3 (TRBV4-3), HD5 (TRBV20-1), whereas no clear enrichment of defined Vβs was detected for HD4, HD6, HD10. HD4 SP14 indicates T cells stimulated with subpool 14 containing peptides 17-18, which elicited the greatest immune response, and HD4 SP18+21 indicates T cells stimulated with subpools 18 and 21, containing peptides 63-64-65-66 and 99-100-101-102, respectively, which elicited the smallest immune response, as shown in Figure 3. For HD7, HD8 and HD9, Vβ immune profiling analysis could not be performed due to reduced cell fitness. [Figure 6-1] Graph showing the results of TCR sequencing of enriched WT1-specific T cells over time. T cells generated from each healthy donor included in the experimental setup were characterized by TCRαβ sequencing after several rounds of stimulation with the WT1 pool. Sequencing results showed the presence of dominant clonotypes for HD1 (a), HD2 (b) and HD3 (c), HD4 (d), HD5 (e), HD6 (f), HD7 (g), HD8 (h), HD9 (i), HD10 (j). Bar graphs depict the 10 most dominant CDR3 amino acid sequences identified at each time point (e.g., S9 corresponds to the sequencing result obtained after the 9th stimulation). For each bar, starting from the x-axis, the lower segment represents the most dominant CDR sequence. The next 9 most dominant sequences are superimposed on the lower section, in order of decreasing frequency going up. The remaining sequences are grouped together in the upper segment. WT1, Wilms tumor 1; CDR3, complementarity determining region 3; S, stimulatory. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3]Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 6-5] Continued from Figure 6-4. [Figure 6-6] Continued from Figure 6-5. [Figure 6-7] Continued from Figure 6-6. [Figure 6-8] Continued from Figure 6-7. [Figure 6-9] Continued from Figure 6-8. [Figure 6-10] Continued from Figure 6-9. [Figure 7]Functional activity of genetically modified T lymphocytes. T cells isolated from PBMCs of healthy individuals were transduced with bidirectional lentiviral vectors encoding the α and β chains of TCRs isolated from HD1 and HD3. As a control, we transduced T cells with a previously published TCR that recognizes the WT1 126-134 (RMFPNAPYL; SEQ ID NO: 255) peptide when expressed by the HLA-A*0201 allele. Transferred (TR) T lymphocytes were co-cultured for 3 days with (a) T2 cells either pulsed or not with WT1 126-134 peptide or with VLDFAPPGA (SEQ ID NO: 157) peptide (effector:target ratio=1:1); (b) K562 cells either wild type (K562) or genetically modified to express HLA-A*0201 allele (effector:target ratio=1:1); (c) three different primary AML blasts selected according to their expression of HLA-A*0201 allele and WT1 antigen (effector:target ratio=5:1). For the co-culture of T2 and K562 cell lines, we included non-transduced T cells as a control. The results showed the ability of each TCR in recognizing the target peptide when presented by the HLA-A*0201 allele (Figure 7a) and the higher ability of HD1 TCR-transduced T cells in mediating specific and almost complete elimination of K562 cells carrying the HLA*A0201 allele compared to HD3 TR T cells. Of note, there was no substantial killing of target cells observed in co-culture of K562 HLA*A0201 cells with WT1 126-134 TR T cells (Figure 7b). These results were further confirmed by the results of co-culture experiments performed using primary AML blasts from three different AML patients as target cells (pAML1 blasts: WT1- / HLA-A*0201+; pAML2 and pAML3 blasts: WT1+ / HLA-A*0201+). In this experimental setting, each individual T cell population was sorted using specific dextramers prior to co-culture with targets to enhance the purity of effector cells.We observed a higher degree of elimination of both pAML blasts carrying the HLA-A*0201 allele upon coculture with HD1 TR T cells, whereas only pAML3-derived blasts were recognized by HD3 T and WT1 126-134 T cells. UT, untransduced; pAML, primary acute myeloid leukemia; TR, transduced; Dx, dextramer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including" or "includes," or "containing" or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, components, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0063] T cell receptor During antigen processing, antigens are degraded intracellularly and then delivered to the cell surface by major histocompatibility complex (MHC) molecules. T cells can recognize this peptide-MHC complex on the surface of antigen-presenting cells. There are two different types of MHC molecules, MHC I and MHC II, each type delivering peptides to the cell surface from a different cellular compartment.

[0064] T cell receptors (TCRs) are molecules found on the surface of T cells that are involved in the recognition of antigens bound to MHC molecules. Naturally occurring TCR heterodimers consist of alpha (α) and beta (β) chains in approximately 95% of T cells, while approximately 5% of T cells have TCRs consisting of gamma (γ) and delta (δ) chains.

[0065] Engagement of the TCR with antigen and MHC leads to the activation of the T lymphocyte on which the TCR is expressed through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules.

[0066] Each chain of a native TCR is a member of the immunoglobulin superfamily and has one N-terminal immunoglobulin (Ig)-variable (V) domain, one Ig-constant (C) domain, a transmembrane / plasma-spanning region, and a short cytoplasmic tail at the C-terminus.

[0067] The variable domains of both the TCR α and β chains have three hypervariable or complementarity determining regions (CDRs). The TCR α or β chains, for example, comprise CDR1, CDR2, and CDR3, in order from amino terminus to carboxy terminus. Generally, CDR3 is the primary CDR involved in recognition of processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, and CDR1 of the beta chain interacts with the C-terminal portion of peptides. CDR2 is believed to recognize MHC molecules.

[0068] The constant domain of the TCR may consist of a short linkage sequence in which cysteine ​​residues form disulfide bonds, creating a link between the two chains.

[0069] The alpha chain of the TCR of the present invention may have a constant domain encoded by the TRAC gene. An exemplary amino acid sequence of the alpha chain constant domain encoded by the TRAC gene is shown below. IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:128)

[0070] The TCR of the present invention may comprise an alpha chain comprising the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto, preferably at least 75% sequence identity thereto.

[0071] The β chain of the TCR of the present invention may have a constant domain encoded by the TRBC1 or TRBC2 gene. An exemplary amino acid sequence of the β chain constant domain encoded by the TRBC1 gene is shown below. DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO:129)

[0072] An exemplary amino acid sequence of the β chain constant domain encoded by the TRBC2 gene is shown below. DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO:130)

[0073] The TCR of the present invention may comprise a β chain comprising the amino acid sequence of SEQ ID NO: 129, SEQ ID NO: 130, or a variant of SEQ ID NO: 129 and 130 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto, preferably at least 75% sequence identity thereto.

[0074] The TCRs of the present invention may contain one or more additional cysteine ​​residues in each of the α and β chains such that the TCR may contain two or more disulfide bonds in the constant domain.

[0075] Due to its structure, the TCR can associate with other molecules such as CD3, which in mammals has three different chains (γ, δ, and ε) and a ζ chain. These accessory molecules have a negatively charged transmembrane domain and are necessary to spread the signal from the TCR to the cell. The CD3- and ζ-chains together with the TCR form what is known as the T cell receptor complex.

[0076] The signal from the T cell complex is enhanced by the simultaneous binding of the MHC molecule by a specific co-receptor. In helper T cells, this co-receptor is CD4 (specific for MHC class II), whereas in cytotoxic T cells, this co-receptor is CD8 (specific for MHC class I). The co-receptor allows a long-term association between the antigen-presenting cell and the T cell and recruits essential intracellular molecules (e.g., LCK) involved in the signal transduction of activated T lymphocytes.

[0077] Thus, as used herein, the term "T cell receptor" (TCR) refers to a molecule capable of recognizing a peptide when presented by an MHC molecule. The molecule may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. A TCR of the invention may be a soluble TCR, for example, in which one or more constant domains have been removed or altered. A TCR of the invention may comprise a constant domain.

[0078] The present invention also provides an α or β chain derived from such a T cell receptor.

[0079] The TCRs of the present invention may be hybrid TCRs, comprising sequences from more than one species. For example, it has surprisingly been found that mouse TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the TCR may comprise human variable regions, and mouse sequences within the constant region.

[0080] A disadvantage of this approach is that the mouse constant sequences may trigger an immune response that leads to rejection of the transferred T cells. However, the conditioning treatments used to prepare patients for adoptive T cell therapy may result in sufficient immunosuppression to allow engraftment of T cells expressing the mouse sequences.

[0081] Complementarity Determining (CDR) Regions The part of the TCR that establishes the majority of contacts with antigenic peptides bound to the major histocompatibility complex (MHC) is the complementarity determining region 3 (CDR3), which is unique to each T cell clone. The CDR3 region arises during somatic rearrangement events that occur in the thymus and involve discontinuous genes belonging to the variable (V), diversity (for D, β and δ chains) and joining (J) genes. Furthermore, random nucleotides inserted / deleted at the rearrangement locus of each TCR chain gene greatly increase the diversity of the highly variable CDR3 sequences. Thus, the frequency of specific CDR3 sequences in a biological sample indicates the abundance of a particular T cell population. The large diversity of the TCR repertoire in healthy humans provides broad protection against a variety of foreign antigens presented by MHC molecules on the surface of antigen-presenting cells. In this regard, theoretically, up to 10 15 It is noteworthy that multiple different TCRs can arise in the thymus.

[0082] T cell receptor diversity is focused on CDR3, the region primarily responsible for antigen recognition.

[0083] The sequences of the CDR3 regions of the TCRs of the invention may be selected from those set out below in Table 1. The TCRs may comprise CDRs comprising or consisting of a CDR3α and CDR3β pair as set out below.

[0084] The CDRs may contain, for example, one, two, or three substitutions, additions, or deletions from a given sequence, provided that the TCR retains the ability to bind to a WT1 peptide when presented by an MHC molecule.

[0085] As used herein, the term "protein" includes single polypeptide chain molecules and multiple polypeptide complexes in which the individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the term "polypeptide" refers to a polymer in which the monomers are amino acids and are linked together through peptide or disulfide bonds.

[0086] Variants, derivatives, analogues, homologues and fragments In addition to the specific proteins and polynucleotides described herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs and fragments thereof.

[0087] In the context of the present invention, a variant of any given sequence is a sequence in which a particular sequence of residues (amino acid or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or alteration of at least one residue present in the naturally occurring protein.

[0088] A variant amino acid sequence of the invention that is referred to as having up to three amino acid substitutions, additions or deletions may have, for example, 1, 2 or 3 amino acid substitutions, additions or deletions.

[0089] The term "derivative" as used herein, in relation to a protein or polypeptide of the invention, includes any substitution, change, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide substantially retains at least one of its intrinsic functions.

[0090] The term "analog" as used herein in the context of a polypeptide or polynucleotide includes any mimetic, that is, a compound that has at least one of the endogenous functions of the polypeptide or polynucleotide that it mimics.

[0091] The proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues, so long as the intrinsic function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine and tyrosine.

[0092] Substitutions may include replacing one amino acid with a similar amino acid (conservative substitution). Similar amino acids are those having side chain moieties with related properties, such as those grouped together as shown below: (i) Basic side chains: lysine (K), arginine (R), histidine (H), (ii) acidic side chains: aspartic acid (D) and glutamic acid (E); (iii) uncharged polar side chains: asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y); or (iv) Nonpolar side chains: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W) and cysteine ​​(C).

[0093] Any amino acid changes should maintain the ability of the TCR to bind to the WT1 peptide presented by the MHC molecule.

[0094] The variant sequences may include amino acid substitutions, additions, deletions and / or insertions. The changes may be localized in one or more regions of the α or β chains, such as the constant regions, linker, or framework regions, or may be spread throughout the TCR molecule.

[0095] Conservative substitutions, additions or deletions may be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

[0096] TIFF2025072432000001.tif55101

[0097] The invention also encompasses homologous substitutions (both substitution and replacement are used herein to mean the replacement of an existing amino acid residue with an alternative residue), e.g., equivalent substitutions, e.g., basic to basic, acidic to acidic, polar to polar, etc. Non-homologous substitutions may also occur, e.g., from one class of residue to another, or may involve the inclusion of an unnatural amino acid, e.g., ornithine.

[0098] The term "variant" as used herein may refer to an entity that has a certain homology to a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" may be considered synonymous with "identity."

[0099] A variant sequence may comprise an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, preferably at least 95%, at least 97%, or at least 99% identical. Typically, the variant comprises the same active site etc. as the subject amino acid sequence. Although homology may also be considered in the context of the present invention in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), it is preferred to express homology in terms of sequence identity.

[0100] A variant sequence may comprise a nucleotide sequence which may be at least 40%, 45%, 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, preferably at least 95%, at least 97%, or at least 99% identical. Although homology may also be considered in the context of the present invention in terms of similarity, it is preferred to express homology in terms of sequence identity.

[0101] Preferably, reference to a sequence having a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence having the given percent identity over the entire length of the referenced SEQ ID NO.

[0102] Identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0103] Percentage homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence, and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0104] This is a very simple and consistent method, but it fails to take into account that, for example, in an otherwise identical pair of sequences, a single insertion or deletion in a nucleotide sequence may exclude the subsequent codon from the alignment, thereby resulting in a large reduction in the percent homology when a global alignment is performed. Most sequence comparison methods are therefore designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0105] However, these more sophisticated methods assign a "gap penalty" to each gap that occurs during alignment, so that a sequence alignment with as few gaps as possible, reflecting a higher relatedness between the two compared sequences, for the same number of identical amino acids, will achieve a higher score than one with many gaps. An "affine gap cost" is typically used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties naturally result in optimized alignments that contain fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparison, it is preferred to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0106] Therefore, calculation of maximum percentage homology first requires the generation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., (1984), Nucleic Acids Res. 12:387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., (1999), supra, Chapter 18), FASTA (Atschul et al., (1990), J. Mol. Biol., pp. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., (1999), supra, pp. 7-58 to 7-60). However, for some applications it is preferred to use the GCG Bestfit program. Another tool called BLAST2 sequence analysis is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999), 174:247-50; FEMS Microbiol. Lett. (1999), 177:187-8).

[0107] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values ​​or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values ​​for the GCG package, or in the case of other software, the default matrix, e.g., BLOSUM62.

[0108] Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0109] A "fragment" is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0110] Such variants can be prepared using standard recombinant DNA techniques, for example, site-directed mutagenesis. If an insertion is to be made, a synthetic DNA can be made that encodes the insert, together with 5' and 3' flanking regions that correspond to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction enzyme sites that correspond to sites in the naturally occurring sequence, so that the sequence can be cleaved with an appropriate enzyme(s) and the synthetic DNA ligated to the cleavage. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for the manipulation of DNA sequences, and other known techniques can also be used.

[0111] major histocompatibility complex (MHC) molecules Typically, the TCR binds a peptide as part of a peptide:MHC complex.

[0112] The MHC molecule may be an MHC class I or II molecule. The complex may be on the surface of an antigen presenting cell, such as a dendritic cell or B cell, or any other cell, including a cancer cell, or it may be immobilized, for example by coating on a bead or plate.

[0113] Human leukocyte antigen system (HLA) is the name of the gene complex that codes for the major histocompatibility complex (MHC) in humans, which includes HLA class I antigens (A, B, and C) and HLA class II antigens (DP, DQ, and DR). HLA alleles A, B, and C present peptides that are primarily derived from intracellular proteins, e.g., proteins expressed inside cells. This is particularly relevant since WT1 is an intracellular protein.

[0114] During T cell development in vivo, T cells undergo a positive selection process to ensure that they recognize self-MHC, followed by a negative process to remove T cells that bind too tightly to MHC presenting self-antigens. As a result, certain T cells, and the TCRs they express, will only recognize peptides presented by certain types of MHC molecules, i.e., those encoded by specific HLA alleles. This is known as HLA restriction.

[0115] One HLA allele of interest is HLA-A * 0201, which is expressed in the vast majority (>50%) of the Caucasian population. * Presented by MHC encoded by HLA-A * TCRs that bind to WT1 peptides (which are .OMEGA.0201-restricted) are advantageous because immunotherapy using such TCRs is suitable for treating a large portion of the Caucasian population.

[0116] Other HLA-A alleles of interest are HLA-A * 0101, HLA-A * 2402 and HLA-A * It's 0301.

[0117] The widely expressed HLA-B allele of interest is HLA-B * 3501, HLA-B * 0702 and HLA-B * The number is 3502.

[0118] The TCR of the present invention is HLA-A * 0201 may be restrictive.

[0119] In one aspect, when the TCR of the present invention comprises a CDR3α having an amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0120] In another aspect, when the TCR of the present invention comprises a CDR3α having an amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO: 41), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0121] In another aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0122] In another aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0123] In another aspect, when the TCR of the present invention comprises a CDR3α having an amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0124] In another aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0125] In another aspect, when the TCR of the present invention comprises a CDR3α having an amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0126] In another aspect, when the TCR of the present invention comprises a CDR3α having an amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0127] In another aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0128] In another aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0129] In another aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-A * 0201 is restrictive.

[0130] In one embodiment, HLA-A * The TCR of the present invention that is 0201-restricted binds to a WT1 peptide comprising the amino acid sequence APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0131] In one aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), comprising a CDR3α that comprises the amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0132] In another aspect, the present invention provides a TCR that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), comprising a CDR3α that comprises the amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO: 41), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0133] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by a major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to an HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0134] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0135] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAASGGRDDKIIF (SEQ ID NO:214), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO:219), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR being linked to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0136] In another aspect, the invention provides a TCR that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by a major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to an HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0137] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR being linked to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0138] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAASATGNQFYF (SEQ ID NO:266), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASSPGQHGELFF (SEQ ID NO:271), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR being linked to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0139] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0140] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by a major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO:277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO:282), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to an HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0141] In another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to HLA-A * The TCR is 0201-restricted, and the WT1 peptide comprises the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0142] Other widely expressed HLA alleles of interest are HLA-B * The TCR of the present invention is HLA-B * 3501 may be restrictive.

[0143] In one aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO: 80), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-B * 3501 is restrictive.

[0144] Thus, in another aspect, the invention provides a TCR that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO: 80), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR being associated with HLA-B * The TCR is 3501-restricted, and the WT1 peptide comprises the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0145] In one aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO: 86), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-B * 3501 is restrictive.

[0146] Thus, in another aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO: 86), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR binding to HLA-B *The TCR is 3501-restricted, and the WT1 peptide comprises the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0147] Other widely expressed HLA alleles of interest are HLA-B * The TCR of the present invention is HLA-B 3502. * 3502 may be restrictive.

[0148] In one aspect, when the TCR of the present invention comprises a CDR3α having the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO: 57), or a variant thereof having up to three amino acid substitutions, additions or deletions, the TCR is HLA-B * 3502 is restrictive.

[0149] Thus, in one aspect, the invention provides a TCR that binds to a Wilms Tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), said TCR comprising a CDR3α that comprises the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β that comprises the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO: 57), or a variant thereof having up to three amino acid substitutions, additions or deletions, said TCR being associated with HLA-B * The TCR is 3502-restricted, and the WT1 peptide comprises the amino acid sequence EPASQHTLRSG (SEQ ID NO: 123), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0150] The present inventors have found that T cells expressing the TCR of the present invention that binds to the WT1 peptide containing the amino acid sequence of EPASQHTLRSG (SEQ ID NO: 123) are related to HLA-B *It has been demonstrated that cancer (AML) cells expressing the 3502 allele can be selectively eliminated - see Example 4 and Figure 4c.

[0151] In one aspect, when the TCR of the present invention binds to a WT1 peptide comprising the amino acid sequence of EPASQHTLRSG (SEQ ID NO: 123), or a variant thereof having up to three amino acid substitutions, additions, or deletions, the TCR is HLA-B * 3502 is restrictive.

[0152] In one embodiment, when the TCR of the present invention binds to a WT1 peptide comprising the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions, the TCR is an HLA-A * 0201 is restrictive.

[0153] In one embodiment, when the TCR of the present invention binds to a WT1 peptide comprising the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127), or a variant thereof having up to three amino acid substitutions, additions, or deletions, the TCR is HLA-B * 3501 is restrictive.

[0154] Wilms Tumor 1 (WT1) Protein Wilms' tumor 1 (WT1) is an intracellular protein that encodes a zinc finger transcription factor that plays an important role in cell growth and differentiation (Yang, L. et al. Leukemia 21, 868-876 (2007)). It is widely expressed on various hematological and solid tumors, but shows limited expression on other tissues (gonads, uterus, kidney, mesothelium, progenitor cells in various tissues). Recent evidence suggests that WT1 is involved in leukemogenesis and tumorigenesis.

[0155] WT1 has several isoforms, some of which arise from alternative splicing of the mRNA transcript encoding WT1. The complete amino acid sequence of one WT1 isoform was previously published (Gessler, M. et al. Nature; 343(6260):774-778; (1990)). This particular isoform consists of 575 amino acids, including the first 126 amino acids at the N-terminus, which are missing from the exon 5+ and KTS+ isoforms of WT1.

[0156] An exemplary WT1 protein has the amino acid sequence set forth in UniProt entry J3KNN9. Other exemplary WT1 proteins have the amino acid sequences set forth below: TIFF2025072432000002.tif52162

[0157] WT1 peptide As used herein, the term peptide refers to multiple amino acid residues linked by peptide bonds. As defined herein, a peptide may be less than about 30, less than about 25, less than about 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 amino acid residues in length. Preferably, the peptide is about 5-20 amino acids in length, more preferably the peptide is about 8-15 amino acid residues in length.

[0158] The TCR of the present invention binds to a WT1 peptide when it is presented by MHC. As used herein, the term WT1 peptide is understood to mean a peptide comprising an amino acid sequence derived from the WT1 protein.

[0159] For example, a WT1 peptide can comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 25 consecutive amino acid residues of a WT1 protein amino acid sequence.

[0160] The WT1 peptide may comprise or consist of the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions, or deletions. Examples of WT1 peptides comprising the amino acid sequence are AAQWAPVLDFAPPGA (SEQ ID NO: 115) and APVLDFAPPGASAYG (SEQ ID NO: 116).

[0161] The WT1 peptide may comprise or consist of an amino acid sequence selected from the group consisting of QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), and variants of SEQ ID NOs: 118-120, each having up to three amino acid substitutions, additions or deletions.

[0162] A WT1 peptide may comprise or consist of an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO:123), YESDNHTTPIL (SEQ ID NO:126), and variants of SEQ ID NOs:123 and 126 having up to three amino acid substitutions, additions, or deletions, respectively. An exemplary WT1 peptide may have an amino acid sequence selected from the group consisting of TCVPEPASQHTLRSG (SEQ ID NO:121), EPASQHTLRSGPGCL (SEQ ID NO:122), HSTGYESDNHTTPIL (SEQ ID NO:124), and YESDNHTTPILCGAQ (SEQ ID NO:125).

[0163] The WT1 peptide may comprise or consist of the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0164] The WT1 peptide may comprise or consist of the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250), or a variant thereof having up to three amino acid substitutions, additions, or deletions. An exemplary WT1 peptide may have an amino acid sequence selected from the group consisting of CMTWNQMNLGATLKG (SEQ ID NO: 248) and NQMNLGATLKGVAAG (SEQ ID NO: 249).

[0165] The WT1 peptide may comprise or consist of the amino acid sequence of DPGGIWAKLGAAEAS (SEQ ID NO: 251), or a variant thereof having up to three amino acid substitutions, additions, or deletions.

[0166] The WT1 peptide may comprise, or consist of, an amino acid sequence selected from the group consisting of NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254), and variants of each of SEQ ID NOs: 252, 253 and 254 having up to three amino acid substitutions, additions or deletions.

[0167] In some embodiments, HLA-A *For a WT1 peptide that binds to an MHC molecule encoded by the 0201 allele, the amino acid at position 2 of the peptide (i.e., the second amino acid from the N-terminus) may preferably be leucine or methionine, although isoleucine, valine, alanine, and threonine may also be preferred. The amino acid at position 9 or 10 may preferably be valine, leucine, or isoleucine, although alanine, methionine, and threonine may also be preferred. Preferred MHC-binding motifs for other HLA alleles are disclosed in Celis et al. (Molecular Immunology, Vol. 31, 8, December 1994, pp. 1423-1430).

[0168] Various uses of the WT1 peptides described herein are contemplated by the present invention.For example, the WT1 peptides described herein can be administered to a subject, for example, a human subject.Administration of the WT1 peptides of the present invention can induce an immune response against cells that express or overexpress WT1 protein, i.e., the WT1 peptides are immunogenic WT1 peptides.

[0169] Accordingly, in another aspect, the present invention provides an isolated immunogenic WT1 peptide comprising an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO:123), YESDNHTTPIL (SEQ ID NO:126), NHTTPILCGAQYRIH (SEQ ID NO:127), QCLSAFTVHFSGQFT (SEQ ID NO:118), EDPMGQQGSLGEQQY (SEQ ID NO:119), SQLECMTWNQMNLGA (SEQ ID NO:120), APVLDFAPPGA (SEQ ID NO:117), NQMNLGATLKG (SEQ ID NO:250), DPGGIWAKLGAAEAS (SEQ ID NO:251), NHTTPILCGAQYRIH (SEQ ID NO:252), KRHQRRHTGVKPFQC (SEQ ID NO:253), PSCQKKFARSDELVR (SEQ ID NO:254), and each of these variants having up to three amino acid substitutions, additions, or deletions.

[0170] The WT1 peptides described herein, e.g., EPASQHTLRSG (SEQ ID NO: 123) and WT1 peptides comprising an amino acid sequence selected from the group consisting of YESDNHTTPIL (SEQ ID NO: 126), NHTTPILCGAQYRIH (SEQ ID NO: 127), QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), SQLECMTWNQMNLGA (SEQ ID NO: 120), APVLDFAPPGA (SEQ ID NO: 117), NQMNLGATLKG (SEQ ID NO: 250), DPGGIWAKLGAAEAS (SEQ ID NO: 251), NHTTPILCGAQYRIH (SEQ ID NO: 252), KRHQRRHTGVKPFQC (SEQ ID NO: 253), PSCQKKFARSDELVR (SEQ ID NO: 254), and their respective variants having up to three amino acid substitutions, additions, or deletions, can be used to screen for and / or identify new TCR sequences that bind to WT1 cells. For example, T2 cells can be pulsed with the WT1 peptides referred to in the present invention and incubated with a T cell population isolated from a donor. In this approach, the expression of cytokines such as CD107a and IFNγ can be an indicator of T cells that recognize the WT1 peptide.

[0171] Accordingly, in one aspect, the invention provides a T cell receptor (TCR) that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), wherein the WT1 peptide is selected from the group consisting of EPASQHTLRSG (SEQ ID NO:123), YESDNHTTPIL (SEQ ID NO:126), NHTTPILCGAQYRIH (SEQ ID NO:127), QCLSAFTVHFSGQFT (SEQ ID NO:118), EDPMGQQGSLGEQQY (SEQ ID NO:119), SQLECMTWNQMNLGA (SEQ ID NO:120), APVLDFAPPGA (SEQ ID NO:117), NQMNLGATLKG (SEQ ID NO:250), DPGGIWAKLGAAEAS (SEQ ID NO:251), NHTTPILCGAQYRIH (SEQ ID NO:252), KRHQRRHTGVKPFQC (SEQ ID NO:253), PSCQKKFARSDELVR (SEQ ID NO: 254), and respective variants thereof having up to three amino acid substitutions, additions or deletions.

[0172] TCR sequence The present inventors have determined the amino acid sequences of TCRs that bind to the WT1 peptides described herein, in particular, the amino acid sequences of TCR CDRs that are important for WT1 peptide recognition and binding.

[0173] Thus, in one embodiment, the present invention provides a TCR comprising a CDR3α having an amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0174] Thus, in one embodiment, the present invention provides a TCR comprising a CDR3α having an amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO: 41), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0175] Thus, in one embodiment, the present invention provides a TCR comprising a CDR3α having an amino acid sequence of CAANNARLMF (SEQ ID NO: 92) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97) or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117) or a variant thereof having up to three amino acid substitutions, additions or deletions when it is presented by MHC.

[0176] Thus, in one embodiment, the present invention provides a TCR comprising a CDR3α having the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0177] In one embodiment, the present invention provides a TCR comprising a CDR3α having an amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0178] In one embodiment, the present invention provides a TCR comprising a CDR3α having an amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0179] Thus, in one embodiment, the present invention provides a TCR comprising a CDR3α having an amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0180] The present invention also relates to a method for producing a pharmaceutical composition comprising the steps of: a CDR3α comprising the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions; a CDR3α comprising the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions; a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions; or a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β comprising the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions; A TCR comprising: The TCR binds to a WT1 peptide comprising or consisting of an amino acid sequence selected from the group consisting of QCLSAFTVHFSGQFT (SEQ ID NO: 118), EDPMGQQGSLGEQQY (SEQ ID NO: 119), and SQLECMTWNQMNLGA (SEQ ID NO: 120), or a variant of each of these having up to three amino acid substitutions, additions, or deletions, when it is presented by MHC.

[0181] Thus, in one embodiment, a TCR is provided that comprises a CDR3α having the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of the amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0182] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0183] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0184] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0185] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0186] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0187] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0188] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0189] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0190] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of QCLSAFTVHFSGQFT (SEQ ID NO: 118), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0191] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of EDPMGQQGSLGEQQY (SEQ ID NO: 119), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0192] In one embodiment, a TCR is provided that comprises a CDR3α having an amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO: 30), or a variant thereof having up to three amino acid substitutions, additions or deletions, and that binds to a WT1 peptide comprising or consisting of an amino acid sequence of SQLECMTWNQMNLGA (SEQ ID NO: 120), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0193] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO: 57), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide comprising or consisting of the amino acid sequence of EPASQHTLRSG (SEQ ID NO: 123), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0194] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO:52), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASTQTPYEQYF (SEQ ID NO:63), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide comprising or consisting of the amino acid sequence of YESDNHTTPIL (SEQ ID NO:126), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0195] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO:52), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSTVGGEDYGYTF (SEQ ID NO:69), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide comprising or consisting of the amino acid sequence of YESDNHTTPIL (SEQ ID NO:126), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0196] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO: 80), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0197] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO: 86), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 127), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0198] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAVTVGNKLVF (SEQ ID NO: 175), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASRGWREQFF (SEQ ID NO: 180), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of DPGGIWAKLGAAEAS (SEQ ID NO: 251), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0199] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSWGYQETQYF (SEQ ID NO: 196), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of NHTTPILCGAQYRIH (SEQ ID NO: 252), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0200] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of KRHQRRHTGVKPFQC (SEQ ID NO: 253), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0201] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of PSCQKKFARSDELVR (SEQ ID NO: 254), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0202] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0203] Further provided by the present invention is a TCR comprising a CDR3α having an amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSDTRAREQFF (SEQ ID NO: 97), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0204] Further provided by the present invention is a TCR comprising a CDR3α having an amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having an amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having an amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0205] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0206] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0207] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0208] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of NQMNLGATLKG (SEQ ID NO: 250), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0209] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0210] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0211] Further provided by the present invention is a TCR comprising a CDR3α having the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3β having the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions, which binds to a WT1 peptide having the amino acid sequence of APVLDFAPPGA (SEQ ID NO: 117), or a variant thereof having up to three amino acid substitutions, additions or deletions, when it is presented by MHC.

[0212] Exemplary TCR amino acid sequences of the invention are provided in Table 1. [Table 1] TIFF2025072432000004.tif251166TIFF2025072432000005.tif253166TIFF2025072432000006.tif251165TIFF202 5072432000007.tif247168TIFF2025072432000008.tif252166TIFF2025072432000009.tif252165TIFF20250724320 00010.tif251166TIFF2025072432000011.tif253166TIFF2025072432000012.tif250165TIFF2025072432000013.t if250166TIFF2025072432000014.tif250165TIFF2025072432000015.tif250165TIFF2025072432000016.tif167164

[0213] Thus, the present invention provides isolated polypeptides comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 114 and 160 to 222, fragments, variants and homologues thereof.

[0214] In one aspect, the invention provides a TCR comprising a TCR α chain sequence selected from the group consisting of the HD1 to HD10 α chain sequences in Table 1, and a TCR β chain sequence independently selected from the group consisting of the HD1 to HD10 β chain sequences in Table 1.

[0215] Reduced mispairing and improved TCR expression The TCRs of the present invention can be expressed in T cells to modify the antigen specificity of the T cells. TCR-transduced T cells can express at least two TCR α chains and two TCR β chains. The endogenous TCR α / β chains form a self-tolerant receptor, whereas the introduced TCR α / β chains form a receptor with a defined specificity for a given target antigen.

[0216] However, TCR gene therapy requires sufficient expression of the introduced TCR, which may be diluted by the presence of endogenous TCR, resulting in suboptimal expression of tumor-specific TCR. Furthermore, when introduced into a patient, mispairing between the endogenous and introduced chains may occur to form new receptors, which may show unexpected specificity against self-antigens and result in autoimmune damage.

[0217] Therefore, several strategies have been explored to reduce the risk of mispairing between endogenous and introduced TCR chains. Mutation of the TCR α / β interface is one strategy currently used to reduce undesired mispairing. For example, the introduction of cysteines into the constant domains of the α and β chains allows disulfide bond formation, enhancing pairing of the introduced chains while reducing mispairing with the wild-type chains.

[0218] Thus, the TCRs of the invention may comprise one or more mutations at the α / β chain interface such that when the α and β chains are expressed in a T cell, the frequency of mispairing between the chains and the endogenous TCR α and β chains is reduced. In one embodiment, the one or more mutations introduce a cysteine ​​residue into the constant region domain of each of the α and β chains, where the cysteine ​​residue is capable of forming a disulfide bond between the α and β chains.

[0219] Another strategy for reducing mispairing relies on the introduction of polynucleotide sequences encoding siRNAs that are appended to genes encoding tumor-specific TCR α and / or β chains and designed to limit expression of endogenous TCR genes (Okamoto S. Cancer research 69, 9003-9011, 2009).

[0220] Thus, vectors or polynucleotides encoding the TCRs of the present invention may comprise one or more siRNAs or other agents aimed at limiting or suppressing expression of endogenous TCR genes.

[0221] It is also possible to combine artificial nucleases, such as zinc finger nucleases (ZFN), transcription activation-like effector nucleases (TALEN) or CRISPR / Cas systems, designed to target endogenous genes, such as the constant regions of TCR genes (TRAC and / or TRBC), to obtain permanent disruption of endogenous TCR α and / or β chain genes, thus allowing full expression of tumor-specific TCRs and thus reducing or eliminating the risk of TCR mispairing. This process, known as TCR gene editing, has shown superiority over TCR gene transfer in vitro and in vivo (Provasi E., Genovese P., Nature Medicine May; 18(5):807-15; 2012).

[0222] Thus, the TCRs of the present invention may be used to edit the specificity of T cells by TCR disruption and genetic addition of tumor-specific TCRs.

[0223] Furthermore, genome editing techniques allow the targeted integration of an expression cassette comprising a polynucleotide encoding the TCR of the invention, and optionally one or more promoter regions and / or other expression control sequences, into endogenous genes that have been disrupted by artificial nucleases (Lombardo A., Nature biotechnology 25, 1298-1306; 2007).

[0224] Thus, the TCRs of the invention may be used to edit T cell specificity by targeted integration of a polynucleotide encoding a TCR of the invention in a genomic region, which integration may be targeted by an artificial nuclease.

[0225] Another strategy developed to increase the expression of the introduced TCR and reduce TCR mispairing consists in "murinization", which replaces the human TCR α and TCR β constant regions (e.g., TRAC, TRBC1 and TRBC2 regions) with their murine counterparts. Murinization of TCR constant regions is described, for example, in Sommermeyer and Uckert J Immunol; 2010 (184:6223-6231). Thus, the TCRs of the present invention can be murinized.

[0226] Isolated Polynucleotides The present invention relates to isolated polynucleotides encoding the TCR receptor of the present invention or parts thereof, such as the α chain and / or β chain, the variable domain or parts thereof.

[0227] The isolated polynucleotide can be double-stranded or single-stranded and can be RNA or DNA.

[0228] Those skilled in the art will understand that many different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. It should further be understood that those skilled in the art can, using routine techniques, make nucleotide substitutions, additions, or deletions which do not affect the polypeptide sequence encoded by a polynucleotide of the invention to reflect the codon usage of any particular host organism in which the polypeptide of the invention is expressed.

[0229] The polynucleotides described herein may be modified by any method available in the art. Such modifications may be made in order to enhance the in vivo activity or life span of polynucleotides of the invention.

[0230] A polynucleotide, such as a DNA polynucleotide, may be produced recombinantly, synthetically, or by any means available to those of skill in the art. It may also be cloned by standard techniques.

[0231] Longer polynucleotides may generally be produced using recombinant methods, for example, using polymerase chain reaction (PCR) cloning techniques. This would involve creating a primer pair (e.g., about 15-30 nucleotides) that flank the target sequence desired to be cloned, contacting the primers with mRNA or cDNA obtained from an animal or human cell, performing the polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

[0232] Examples of nucleotide sequences encoding TCRs according to the invention are provided in Table 2.

[0233] [Table 2] TIFF2025072432000018.tif250167TIFF2025072432000019.tif251168TIFF2025072432000020.tif249168TIFF202 5072432000021.tif252168TIFF2025072432000022.tif248167TIFF2025072432000023.tif250166TIFF20250724320 00024.tif251168TIFF2025072432000025.tif250167TIFF2025072432000026.tif251168TIFF2025072432000027.t if250167TIFF2025072432000028.tif249168TIFF2025072432000029.tif252167TIFF2025072432000030.tif114168

[0234] Thus, the present invention provides isolated polynucleotides comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 132-156, 223-247 and 292-299, or variants thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0235] The present invention also provides a TCR comprising an alpha chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 132, 135, 138, 141, 144, 147, 152, 223, 226, 227, 228, 233, 236, 237, 238, 245, 292, 295, and variants thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0236] The present invention also relates to SEQ ID NOs: 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 150, 151, 153, 154, 155, 156, 224, 225, 229, 230, 231, 232, 234, 235, 239, 240, 241, 242, 243, 244, 246, 247, 293, 294, 296-299, and at least one corresponding thereto. The present invention provides a TCR comprising a β chain encoded by a nucleotide sequence selected from the group consisting of variants thereof having 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0237] The present invention further provides isolated polynucleotide sequences derived from the sequences present in Table 2. For example, the present invention provides an isolated polynucleotide encoding a variable region of a TCR according to the present invention, the isolated polynucleotide comprising a stretch of any one of nucleotides set forth in SEQ ID NOs: 132-156, 223-247, and 292-299.

[0238] The variant sequence may have additions, deletions or substitutions of one or more bases. If the changes involve addition(s) or deletion(s), they may either occur in triplicates or be balanced (i.e., an addition for each deletion) such that the changes do not cause a frameshift in translation of the remainder of the sequence.

[0239] Some or all of the changes may be "silent," in the sense that they do not affect the sequence of the encoded protein, due to the degeneracy of the genetic code.

[0240] Some or all of the changes may result in conservative amino acid substitutions, additions, or deletions, as explained above. The changes may be concentrated in one or more regions, such as the regions encoding the constant regions, linker, or framework regions of the α or β chains, or they may be spread throughout the molecule.

[0241] The variant sequence should retain the ability to encode all or a portion of the TCR amino acid sequence that binds to the WT1 peptide.

[0242] Codon Optimization The polynucleotides used in the present invention may be codon-optimized. Codon optimization has already been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their use of certain codons. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in a cell type. By changing the codons in the sequence so that they match the relative abundance of the corresponding tRNA, it is possible to increase expression. Similarly, it is possible to decrease expression by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. Thus, an additional degree of translational control is available.

[0243] Many viruses, including HIV and other lentiviruses, use a number of rare codons, and by changing these to correspond to commonly used mammalian codons, increased expression of the packaging components in mammalian producer cells can be achieved. Codon usage tables for mammalian cells, as well as for a variety of other organisms, are known in the art.

[0244] Codon optimization may also include removal of mRNA instability motifs and cryptic splice sites.

[0245] vector The present invention provides vectors comprising the polynucleotides described herein.

[0246] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the present invention and for purposes of illustration, some vectors used in recombinant nucleic acid technology allow an entity, such as a segment of nucleic acid (e.g., a heterologous DNA segment, e.g., a heterologous cDNA segment), to be transferred into a target cell. A vector may serve the purpose of maintaining a heterologous nucleic acid (DNA or RNA) in a cell, facilitating the replication of a vector containing a segment of nucleic acid, or facilitating the expression of a protein encoded by a segment of nucleic acid. A vector may be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, chromosomes, artificial chromosomes, and viruses. A vector may be single-stranded or double-stranded. It may be linear, and in some cases, the vector comprises one or more homology arms. A vector may also be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, a vector may itself be the nucleotide of interest.

[0247] The vector used in the present invention may be, for example, a plasmid or viral vector and may comprise a promoter for the expression of the polynucleotide and optionally a regulator of the promoter.

[0248] The vectors containing the polynucleotides used in the present invention may be introduced into cells using various techniques known in the art, such as transformation, transfection and transduction. Several techniques are known in the art, such as transduction with recombinant viral vectors, such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses and herpes simplex virus vectors, Sleeping Beauty vectors, direct injection of nucleic acids and biolistic transformation.

[0249] Non-viral delivery systems include, but are not limited to, DNA transfection methods.Thus, transfection includes the process of using non-viral vectors to deliver genes to target cells.Exemplary transfection methods include electroporation, DNA microprojectile bombardment, lipid-mediated transfection, small DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nature Biotechnology, 1996, 14; 556) and combinations thereof.

[0250] The term "transfection" should be understood to encompass delivery of a polynucleotide into a cell by both viral and non-viral delivery.

[0251] In addition, the present invention may utilize gene targeting protocols, such as the delivery of DNA modifying factors.

[0252] The term "vector" includes expression vectors, i.e., constructs capable of in vivo or in vitro / ex vivo expression. Expression may be controlled by vector sequences or, for example, in the case of insertion at a target site, expression may be controlled by target sequences. A vector may be integrated or tethered into the DNA of a cell.

[0253] Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.

[0254] Retroviruses are RNA viruses with a life cycle that is different from that of lytic viruses. In this context, retroviruses are infectious entities that replicate through a DNA intermediate. When a retrovirus infects a cell, its genome is converted into a DNA form by the enzyme reverse transcriptase. The DNA copy serves as a template for the production of a new RNA genome, and virally encoded proteins are required for the assembly of infectious viral particles.

[0255] Many retroviruses exist, such as murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus 29 (MC29), and avian erythroblastosis virus (AEV), and all other retroviruses including lentiviruses.

[0256] A detailed list of retroviruses can be found in Coffin et al. (Retroviruses, 1997, Cold Spring Harbour Laboratory Press, eds. JM Coffin, SM Hughes, HE Varmus, pp. 758-763).

[0257] Lentiviruses also belong to the retrovirus family, but they are able to infect both dividing and non-dividing cells (Lewis et al., (1992), EMBO J, 3053-3058).

[0258] The vector can be capable of transferring the nucleotide sequence encoding the WT1-specific TCR described herein into cells, for example, T cells, so that the cells express the WT1-specific TCR.Preferably, the vector is capable of persistently expressing at high levels in T cells, so that the introduced TCR can successfully compete with endogenous TCR for a limited pool of CD3 molecules.

[0259] As the supply of CD3 molecules increases, TCR expression may increase, for example, in cells modified to express the TCR of the present invention. Thus, the vector of the present invention may further comprise one or more genes encoding CD3-gamma, CD3-delta, CD3-epsilon and / or CD3-zeta. In one embodiment, the vector of the present invention comprises a gene encoding CD3-zeta. The vector may comprise a gene encoding CD8. The vector may encode a selection marker or suicide gene to increase the safety profile of the genetically engineered cells, for example, the cells of the present invention or cells modified to express the TCR of the present invention (Bonini, Science, 1997; Ciceri, Bonini Lancet Oncol., 2009; Oliveira et al., STM, 2015). The genes contained in the vector of the present invention may be bounded by a self-cleaving sequence, for example, a 2A self-cleaving sequence.

[0260] Alternatively, one or more separate vectors encoding a CD3 gene may be provided for co-transfection into a cell simultaneously, sequentially or separately with one or more vectors of the invention, e.g., one or more vectors encoding a TCR of the invention.

[0261] cell The present invention relates to a cell comprising a polynucleotide or a vector according to the invention.

[0262] The cell may be a T cell, lymphocyte, or stem cell. The T cell, lymphocyte, or stem cell may be selected from the group consisting of CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells, and pluripotent stem cells.

[0263] The type of cell may be selected to provide desired and advantageous in vivo persistence, as well as to provide desired and advantageous functions and characteristics for the cells of the invention.

[0264] The cells may be isolated from a subject.

[0265] The cells of the present invention may be provided for use in adoptive cell transfer. As used herein, the term "adoptive cell transfer" refers to the administration of a cell population to a patient. Typically, the cells are T cells isolated from a subject, then genetically modified and cultured in vitro to express the TCR of the present invention before being administered to a patient.

[0266] Adoptive cell transfer may be allogeneic or autologous.

[0267] It should be understood that by "autologous cell transfer", the starting population of cells (which are then transduced by the methods of the invention or with the vectors according to the invention) is obtained from the same subject to which the transduced T cell population is to be administered. Autologous transfer is advantageous because it avoids problems associated with immunological incompatibility and is available to the subject independent of the availability of a genetically matched donor.

[0268] It should be understood that by "allogeneic cell transfer", the starting population of cells (which are then transduced by the methods of the invention or with a vector according to the invention) is obtained from a different subject than the one to which the transduced cell population is to be administered. Preferably, the donor is genetically matched to the subject to which the cells are to be administered to minimize the risk of immunological incompatibility. Alternatively, the donor may be mismatched or unrelated to the patient.

[0269] A suitable dose of the transduced cell population is, for example, a therapeutically and / or prophylactically effective amount. The dose to be administered depends on the subject and condition being treated and can be readily determined by one of skill in the art.

[0270] The cells may be derived from T cells isolated from a subject. The T cells may be part of a mixed cell population isolated from a subject, such as a population of peripheral blood lymphocytes (PBLs). T cells within the PBL population may be activated by methods known in the art, for example, using anti-CD3 and / or anti-CD28 antibodies, or cell-sized beads coupled with anti-CD3 and / or anti-CD28 antibodies.

[0271] T cells are CD4 + Helper T cells or CD8 + The cells may be cytotoxic T cells. + Helper T cells / CD8 + Polyclonal activation, for example, the use of anti-CD3 antibodies, optionally in combination with anti-CD28 antibodies, may be within a mixed population of cytotoxic T cells. + and CD8 + Triggers T cell proliferation.

[0272] Cells may be isolated from the subject into which genetically modified cells should be adoptively transferred. In this regard, cells may be produced by isolating T cells from the subject, optionally activating T cells, and transferring TCR genes to the cells ex vivo.Subsequent immunotherapy of the subject may then be carried out by adoptive transfer of TCR-transduced cells.As used herein, this process refers to autologous T cell transfer.That is, TCR-transduced cells are administered to the same subject from which the T cells were originally derived.

[0273] Alternatively, the T cells may be isolated from different subjects so that they are allogeneic. The T cells may be isolated from a donor subject. For example, if the subject is undergoing allogeneic hematopoietic stem cell transplantation (Allo-HSCT) or solid organ transplantation or cell transplantation or stem cell therapy, the cells may be from the donor from which the organ, tissue or cells are derived. The donor and the subject undergoing treatment may be siblings.

[0274] Alternatively, the cell may be or be derived from a stem cell, such as a hematopoietic stem cell (HSC). Gene transfer into HSC does not lead to TCR expression on the cell surface, since stem cells do not express CD3 molecules. However, when the stem cell differentiates into lymphoid precursors that migrate to the thymus, CD3 expression begins, leading to the surface expression of the introduced TCR on thymocytes.

[0275] The advantage of this approach is that expression of the introduced TCR chains suppresses rearrangement of endogenous TCR gene segments to form functional TCR alpha and beta genes, so that mature T cells, when generated, express only the introduced TCR and little or no endogenous TCR chains. A further advantage is that the genetically modified stem cells are a continuous source of mature T cells with the desired antigen specificity. Thus, the cells may be genetically modified stem cells, preferably genetically modified hematopoietic stem cells, which after differentiation give rise to T cells expressing the TCR of the invention.

[0276] Other approaches known in the art can be used to reduce, limit, prevent, silence or abolish expression of endogenous genes in the cells of the invention or in cells prepared by the methods of the invention.

[0277] As used herein, the term "disrupting" refers to reducing, restricting, preventing, silencing, or suppressing the expression of a gene. A person skilled in the art can use any suitable method known in the art for disrupting an endogenous gene, such as genome editing, gene silencing, gene knockdown, or gene knockout.

[0278] For example, endogenous genes can be destroyed using artificial nucleases.Artificial nucleases are, for example, artificial restriction enzymes that are engineered to selectively target specific polynucleotide sequences (e.g., encoding genes of interest) and induce double-strand breaks in the polynucleotide sequences.Typically, double-strand breaks (DSBs) are repaired by error-prone non-homologous end joining (NHEJ), which results in the formation of non-functional polynucleotide sequences that may not be able to express endogenous genes.

[0279] In some embodiments, the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas (e.g., CRISPR / Cas9).

[0280] The method of preparing the cells (e.g., T cells) of the invention may include a step of targeted integration of an expression cassette into an endogenous gene (e.g., an endogenous TCR α chain gene and / or an endogenous TCR β chain gene). As used herein, the term expression cassette refers to a polynucleotide sequence (e.g., a DNA polynucleotide sequence) comprising one or more polynucleotide sequences encoding one or more genes of interest such that the genes of interest can be expressed. The endogenous sequences may facilitate expression from the expression cassette and / or a transcription control sequence within the expression cassette may facilitate expression. For example, the expression cassette may comprise a polynucleotide sequence of the invention or a polynucleotide sequence encoding a TCR of the invention operably linked to an expression control sequence, e.g., a promoter or enhancer sequence. The one or more genes of interest may be located between one or more sets of restriction enzyme sites. Suitably, the restriction enzyme sites may facilitate integration of the expression cassette into, for example, a vector, a plasmid, or genomic DNA (e.g., host cell genomic DNA).

[0281] For example, an expression cassette of the present invention may be transferred from a first polynucleotide sequence, e.g., on a vector, to another by "cutting out", e.g., excising, the expression cassette using one or more suitable restriction enzymes, and "pasting", e.g., incorporating, the expression cassette into the second polynucleotide sequence.

[0282] The expression cassette may comprise a polynucleotide of the invention. The expression cassette may comprise a polynucleotide encoding one or more TCRs of the invention. The expression cassette may further comprise an antibiotic resistance gene or other selectable marker gene that allows cells that have successfully integrated the expression cassette into their DNA to be identified. The polynucleotide sequence contained in the expression cassette may be operably linked to an expression control sequence, such as a suitable promoter or enhancer sequence. Those skilled in the art can select a suitable expression control sequence.

[0283] The present invention also contemplates cells expressing the TCR of the present invention that have been engineered to disrupt one or more endogenous MHC genes. Disruption of endogenous MHC genes can reduce or prevent the expression of MHC on the engineered cell surface. Thus, such engineered cells with or without reduced MHC expression have limited or no ability to present antigens on their cell surface. Such cells are particularly advantageous for adoptive cell transfer because the cells are non-allo-reactive, e.g., they do not present antigens that can be recognized by the immune system of the subject receiving the adoptively transferred cells. As a result, the transferred cells are not recognized as "non-self" and adverse immune reactions against the cells can be avoided. Such cells are called "universal cells" because they are suitable for adoptive transfer into a variety of different hosts, regardless of HLA type.

[0284] Thus, the present invention provides a method for preparing a non-allo-reactive universal T cell expressing a TCR of the present invention. Further provided by the present invention is a non-allo-reactive universal T cell expressing a TCR of the present invention.

[0285] The present invention further contemplates cells engineered to disrupt one or more endogenous genes to modify the cell and enhance advantageous properties, characteristics or functions of the cell and / or reduce undesirable properties, characteristics or functions. For example, disrupting the endogenous cells may modify persistence, amplification, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other cell functions. As used in this context, the term "modify" refers to a change in one or more characteristics compared to an equivalent unmodified cell, e.g., a cell in which the endogenous genes are not disrupted. For example, the change may be an increase, enhancement, or introduction of a cellular characteristic or function compared to an equivalent unmodified cell. Alternatively, the change may be a decrease, suppression, or abrogation of a cellular characteristic or function compared to an equivalent unmodified cell.

[0286] The polynucleotides and vectors of the invention may be transferred to specific T cell subsets, including CD4 and / or CD8, naive, memory stem T cells, central memory, effector memory or effector cells, or other cell subsets, for example to promote different in vivo persistence and function of the cells of the invention.

[0287] The polynucleotides and vectors of the invention may also be transferred to T cell subsets, such as naive, memory stem T cells, central memory cells, effector memory cells, effector.

[0288] The polynucleotides and vectors of the invention may also be transferred into T cell subsets with different polarizations, e.g., Th0 / Tc0, Th1 / Tc1, Th2 / Tc2, Th17, Th22 or others, depending on the cytokine background most suitable for targeting a particular tumor type.

[0289] Additionally, the polynucleotides and vectors of the present invention encoding antigen-specific regions of the TCRs of the present invention may be transferred to other cell subsets, including gamma / delta T cells, NK cells, NKT cells, hematopoietic stem cells or other cells, to provide a therapeutic effect.

[0290] Further provided by the present invention is a method for preparing a cell, the method comprising transducing the cell in vitro or ex vivo with a vector of the present invention. Various methods for transducing cells with vectors are known in the art (see, e.g., Sambrook et al.).

[0291] The invention also provides a method of generating T cells expressing a TCR of the invention by inducing differentiation of a stem cell containing a polynucleotide or vector of the invention.

[0292] A population of cells may be selectively purified for cells that exhibit a particular phenotype or characteristic, or from other cells that do not exhibit that phenotype or characteristic, or that exhibit it to a lesser extent. For example, a population of cells expressing a particular marker (e.g., CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) can be purified from a starting population of cells. Alternatively, or in addition, a population of cells that does not express another marker can be purified.

[0293] It should be understood that by "enriching" a population of cells for a certain type of cell, the concentration of that type of cell is increased within the population. The concentration of other types of cells may be simultaneously decreased.

[0294] Purification or enrichment can result in a population of cells that is substantially pure from other types of cells.

[0295] Purification or enrichment for a population of cells expressing a particular marker (e.g., CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) can be achieved by using an agent that binds to the marker, preferably that binds substantially specifically to the marker. The agent that binds to the cell marker may be an antibody, for example, an antibody that binds to CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4.

[0296] The term "antibody" refers to intact antibodies or antibody fragments capable of binding to a selected target and including Fv, ScFv, F(ab') and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies, including chimeric, CDR-grafted and humanized antibodies, and artificially selected antibodies produced using phage display or alternative technologies.

[0297] In addition, alternatives to classical antibodies, such as "avibodies", "avimers", "anticalins", "nanobodies" and "DARPins" may also be used in the present invention.

[0298] The agent that binds to a particular marker may be labeled so that it can be identified using any of a number of techniques known in the art. The agent may be inherently labeled or may be modified by conjugating a label to it. By "conjugating", it should be understood that the agent and the label are operatively linked. This means that the agent and the label are linked together in such a way that they can perform both of their functions (e.g., binding to the marker, allowing fluorescent identification, or allowing separation when placed in a magnetic field) without substantial hindrance. Suitable conjugation methods are well known in the art or can be easily identified by those skilled in the art.

[0299] The label may, for example, allow the labeled agent, and any cells to which it binds, to be purified from its environment (e.g., the agent may be labeled with magnetic beads or affinity tags, such as avidin), detected, or both. Detectable markers suitable for use as labels include fluorophores (e.g., green, cherry, cyan and orange fluorescent proteins) and peptide tags (e.g., His tags, Myc tags, FLAG tags and HA tags).

[0300] Numerous techniques are known in the art for isolating populations of cells expressing particular markers, including magnetic bead-based separation techniques (e.g., closed-circuit magnetic bead-based separation), flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g., using affinity columns or beads, e.g., biotin columns to separate avidin-labeled drugs), and microscopy-based techniques.

[0301] It may also be possible to perform the separation using a combination of different techniques, for example a magnetic bead-based separation step followed by sorting of the resulting cell population for one or more additional (positive or negative) markers by flow cytometry.

[0302] Clinical grade separations can be performed, for example, using the CliniMACS® system (Miltenyi), which is an example of a closed-circuit magnetic bead-based separation technology.

[0303] It is also envisioned that dye exclusion properties (eg, side population or rhodamine labeling) or enzymatic activity (eg, ALDH activity) can be used to enrich for HSCs.

[0304] Chimeric molecules In another aspect, the invention provides a chimeric molecule comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, or a portion thereof, linked to non-cellular material. The linkage may be covalent or non-covalent.

[0305] The non-cellular material may be a nanoparticle, an exosome, or any non-cellular material known in the art.

[0306] The chimeric molecules of the present invention may be soluble.

[0307] In another aspect, the invention provides chimeric molecules comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, or a portion thereof, linked to a toxin or an antibody.

[0308] The toxin or antibody may be cytotoxic. The toxin may be a cytotoxic molecule or compound, for example, a radioactive molecule or compound. The TCR portion of the chimeric molecule may confer the ability to recognize cells expressing WT1 protein or peptide. Thus, the chimeric molecule may specifically recognize and / or bind to WT1-expressing tumor cells. Thus, the chimeric molecule of the present invention may provide WT1-targeted delivery of cytotoxic toxins, antibodies and / or compounds.

[0309] WT1-related diseases WT1 is widely expressed in various blood systems and solid tumors, while showing limited expression in various healthy tissues (e.g., gonads, uterus, kidney, mesothelium, progenitor cells in different tissues). The present inventors have identified and determined the amino acid sequence of TCR that recognizes WT1 peptide. Furthermore, the present inventors have demonstrated that T cells expressing the TCR according to the present invention target and kill cells that present WT1 peptide or overexpress WT1 protein.

[0310] Thus, the present invention provides a method for treating and / or preventing a disease associated with WT1 expression, the method comprising the step of administering a TCR, isolated polynucleotide, vector, or cell of the present invention to a subject in need thereof. The present invention also provides a method for treating and / or preventing a disease associated with WT1 expression, the method comprising the step of administering a cell prepared by the method of the present invention to a subject in need thereof.

[0311] The present invention further provides a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell according to the present invention, or a cell prepared by the method of the present invention, for use in treating and / or preventing a disease associated with expression of WT1.

[0312] The term "preventing" is intended to refer to preventing, delaying, impeding, or hindering the onset of a disease. Treatment may, for example, prevent or reduce the likelihood of developing or contracting a disease associated with expression of WT1.

[0313] As used herein, "treating" refers to caring for a subject with a disease in order to ameliorate, cure, or reduce the symptoms of the disease, or to reduce, halt, or slow the progression of the disease.

[0314] The subject may be a human subject. The human subject may be a child. For example, the child may be under 10 years old, under 9 years old, under 8 years old, under 7 years old, under 6 years old, under 5 years old, under 4 years old, under 3 years old, or under 2 years old. The human subject may be an infant.

[0315] A subject may have already been determined to need the TCR, isolated polynucleotide, vector or cell of the present invention, or the cell prepared by the method of the present invention based on the expression of WT1. For example, a subject may have a cell population that shows increased expression of WT1 compared to a healthy control cell population. WT1 expression may be determined using various techniques known in the art, for example, quantitative RT-PCR can be used to determine the amount of WT1 RNA transcript, which is an indicator of WT1 protein expression. Those skilled in the art will understand that WT1 protein expression can be determined by performing Western blot using a commercially available antibody specific to WT1.

[0316] The subject may also have been previously identified as having an alteration (e.g., mutation or deletion) in the WT1 gene. Such an alteration may be hereditary. Thus, the disease associated with expression of WT1 may be a genetic disease. Examples of genetic diseases associated with expression of WT1 include, but are not limited to, WAGR (Wilms tumor-aniridemia-genitourinary dysplasia-retardation) syndrome, Dennis-Drash syndrome (DDS), Frasier syndrome (FS), and genitourinary anomalies (reproductive and urinary system anomalies) syndrome.

[0317] Subjects with a genetic disease associated with expression of WT1 may be at increased risk of developing a proliferative disorder (eg, cancer).

[0318] The disease associated with expression of WT1 may be a proliferative disorder.

[0319] The proliferative disorder may be a hematological malignancy or a solid tumor. The hematological malignancy may be selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

[0320] The solid tumor may be selected from the group consisting of lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial cancer, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial carcinoma, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0321] The disease associated with the expression of WT1 may be selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial carcinoma, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0322] Pharmaceutical Compositions The TCRs of the invention, the polynucleotides of the invention, the vectors of the invention, the cells of the invention, the cells prepared by the methods of the invention, the chimeric molecules of the invention, and the mixed cell populations of the invention may be formulated for administration to a subject with a pharma- ceutically acceptable carrier, diluent or excipient. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline, and optionally containing human serum albumin.

[0323] Handling of cell therapy products is preferably performed in accordance with the FACT-JACIE International Standards for Cell Therapy.

[0324] Treatment method In another aspect, the present invention provides a method for treating and / or preventing a disease associated with expression of WT1, the method comprising the step of administering a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell of the present invention, a cell prepared by a method of the present invention, a chimeric molecule of the present invention, or a mixed cell population of the present invention to a subject in need thereof.

[0325] The subject may be a human subject. The subject may be a non-human animal subject.

[0326] The subject may have a disease associated with WT1 expression. The subject may be at risk of developing a disease associated with WT1 expression. The subject may have already been determined to be at risk of developing a disease associated with WT1 expression. The subject may have an increased risk of developing a disease associated with WT1 expression.

[0327] The increased risk may be determined by genetic screening and / or by examining the subject's family history. The subject may express genetic markers that are indicative of an increased risk of developing a disease associated with expression of WT1.

[0328] Preferably, a person skilled in the art will know genetic risk factors (e.g., genetic markers) associated with an increased risk of developing a disease associated with WT1. A person skilled in the art can use any suitable method or technique known in the art to determine whether a subject has an increased risk of developing a disease associated with expression of WT1.

[0329] The subject may have previously been treated for a disease associated with expression of WT1. The subject may be in remission. The subject may be resistant to chemotherapy. The subject may be resistant to anti-WT1 therapy.

[0330] In one embodiment, the method for treating and / or preventing a disease associated with WT1 expression comprises administering chemotherapy to a subject. The chemotherapy may be administered to a subject simultaneously, sequentially, or separately with the TCR of the present invention, the isolated polynucleotide of the present invention, the vector of the present invention, the cell according to the present invention, the cell prepared by the method of the present invention, or the chimeric molecule of the present invention.

[0331] In another aspect, the present invention provides a method for treating and / or preventing a disease associated with expression of WT1, comprising the step of administering a mixed cell population, wherein the mixed cell population comprises multiple cell populations, each expressing a different TCR of the present invention.

[0332] In another aspect, the invention provides a mixed cell population comprising multiple cell populations, each expressing a different TCR of the invention.

[0333] In another aspect, the invention provides a method for preparing a mixed cell population comprising multiple cell populations, each expressing a different TCR of the invention, comprising transducing cells in vitro or ex vivo with a vector of the invention.

[0334] In another aspect, the present invention provides a mixed cell population for use in the treatment and / or prevention of a disease associated with expression of WT1, the mixed cell population comprising multiple cell populations, each expressing a different TCR of the present invention.

[0335] For example, a mixed cell population may comprise a first cell population expressing a first TCR of the invention and a second cell population expressing a second TCR of the invention. For example, a mixed cell population may comprise a first cell population expressing a first TCR of the invention, a second cell population expressing a second TCR of the invention, and a third cell population expressing a third TCR of the invention, etc.

[0336] Each cell population of the mixed cell population may express, for example, only one TCR of the present invention. The endogenous TCR gene of the cell population in the mixed cell population may be disrupted or deleted. The expression of the endogenous TCR gene of the cells in the mixed cell population may be disrupted, for example, by gene editing using an artificial nuclease.

[0337] In another aspect, the present invention provides use of a TCR of the present invention, an isolated polynucleotide of the present invention, a vector of the present invention, a cell of the present invention, a cell prepared by a method of the present invention, a chimeric molecule of the present invention, or a mixed cell population of the present invention for the manufacture of a medicament for the treatment of a disease associated with expression of WT1.

[0338] Both human and animal treatments are within the scope of the present invention.

[0339] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, histology, immunology, oncology, which are within the capabilities of those of ordinary skill in the art. Such techniques are explained in the literature.

[0340] See, e.g., Sambrook, J., Fritsch, EF, and Maniatis, T., (1989), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM et al., (1995 and periodic supplements), Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A., (1996), DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM, and McGee, J.O'D., (1990), In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ, (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM, and Dahlberg, See, JE, (1992), Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general textbooks is incorporated herein by reference.

[0341] Various preferred features and embodiments of the present invention will now be described, by way of non-limiting example. EXAMPLES

[0342] Example 1: Generation of functional WT1-specific cytotoxic T lymphocytes (CTLs) from healthy donors (HD) To identify novel TCRs specific for WT1 epitopes restricted by different HLA alleles, we stimulated peripheral blood mononuclear cells (PBMCs) from 10 different HD patients with a pool of pentadecapeptides (15mers) with 11 amino acid overlaps spanning the complete sequence of the WT1 protein (see Materials and Methods). + and CD8 + This ensures optimal stimulation of both T cells.

[0343] After 26-30 hours of stimulation, we enriched for T cells expressing CD137, a molecule that is upregulated upon T cell receptor engagement and is responsible for antigen-specific memory and naive CD4 + and CD8 + It has been shown to be a reliable marker for the rapid identification, isolation and in vitro expansion of T cells. The CD137 negative fraction was further depleted of CD3 and then irradiated with 30 Gy to remove CD137. + The fraction was used as antigen-presenting cells (APCs). + Cells were expanded in vitro for about 9 days and restimulated every 7-14 days with autologous APCs loaded with peptide pools, presented by CD3-depleted cells or by immortalized autologous B cells. This procedure resulted in enrichment of WT1-specific T lymphocytes, as shown by the cytofluorometric results presented in Figure 1(aj). Functional characterization of T cells was performed at different time points. More specifically, T cells were co-cultured with autologous APCs loaded with peptide pools, and after 6 hours of co-culture, expression of CD107a and IFNγ in the T cell population was identified by intracellular staining. Expression of CD107a after antigen encounter indicates antigen-induced degranulation and lysis capacity.

[0344] As a negative control, cells were stimulated with an irrelevant peptide pool, which resulted in minimal secretion of IFNγ and CD107a by T cells from each healthy donor.

[0345] [Example 2] Mapping of WT1 epitopes that induce T cell responses To identify WT1 epitopes recognized by T cells, IFNγ secretion was quantified after 6 h in vitro co-culture of WT1 stimulated / enriched T cells with autologous APCs loaded with peptide subpools, each containing up to 12 peptides, according to a mapping grid. The mapping grid consists of 24 subpools, with each peptide uniquely contained in two intersecting subpools (Doubrovina, E. et al., Blood, 120, 1633-1646, (2012)). The results are outlined in Figure 2a.

[0346] FACS analysis showed substantial expression of IFNγ and CD107a by T cells from HD1, HD3, HD6, HD7, HD10 after stimulation with subpools 4, 5 and 16 (Fig. 2b, d, g, h, k). Substantial expression of IFNγ was observed in T cells from HD2 stimulated with subpools 6, 16, 17 and 20 (Fig. 2c), whereas subpools 4, 5, 6, 14, 18, 21 stimulated expression of IFNγ and CD107a by T cells from HD4 (Fig. 2e) and subpools 5, 11, 12, 21, 22 stimulated expression of IFNγ in HD5 (Fig. 2f). For HD7, we additionally observed increased expression of IFNγ and CD107a after stimulation with subpools 7, 8, 20, albeit at lower levels compared to those observed with subpools 4, 5 and 16 (Fig. 2h). Furthermore, we observed increased expression of IFNγ and CD107a following stimulation of HD8-derived T cells with subpools 12 and 14 (Fig. 2i) and HD9-derived T cells with subpools 5, 13, 21 (Fig. 2j).

[0347] HD-derived T cells were then stimulated for 6 h with APCs pulsed with a single pentadecapeptide that elicits maximal immune responses and is shared by subpools bearing at least one unrelated 15mer. FACS analysis showed increased expression of CD107a and / or IFNγ for peptides 40 and 41 in HD1 T cells (Figure 3a), for peptides 54, 77, 90 for HD2 T cells (Figure 3b), for peptide VLDFAPPGA (SEQ ID NO: 157; which is a nonamer of the peptide represented by SEQ ID NO: 117) for HD3 T cells (Figure 3c), for peptides 17, 18, 99, 100 for HD4 (Figure 3d, e), for peptide 101 for HD5 (Figure 3f), for peptide VLDFAPPGA (SEQ ID NO: 157; which is a nonamer of the peptide represented by SEQ ID NO: 117) for HD6 T cells (Figure 3g), for peptides 101, 125 and 137 for HD9 T cells (Figure 3h), and for peptide VLDFAPPGA (SEQ ID NO: 157; which is a nonamer of the peptide represented by SEQ ID NO: 117) for HD10 T cells (Figure 3i). In this way, dominant immunogenic sequences were identified. No relevant immune response (i.e. increased expression of CD107a and / or IFNγ) was observed after co-culture with an irrelevant control peptide. For HD7 and HD8, due to reduced cell fitness, no culture experiments could be performed to identify immunogenic peptides. Nevertheless, we were able to predict the recognized peptides by deconvolution of the mapping grid. For HD7, we identified overlapping sequences of peptides 41 and 42 (originating from SP4, 5, 16) and peptides 91 and 92 (originating from SP7, 8, 20), and for HD8, peptide 24.

[0348] To determine the HLA restriction of the WT1 immunogenic peptides recognized by T cells expanded from HD4, HD5, and HD10, T cells derived from these were co-cultured for 6 hours with a panel of different target EBV-BLCL cells, each expressing a different HLA-A or HLA-B allele, pulsed with the relevant peptide (peptide 17 for HD4; peptide 101 for HD5; peptide VLDFAPPGA (SEQ ID NO: 157) for HD10) or an irrelevant control peptide. The results of this experiment showed that peptide 17 was specifically expressed by HLA-B * Peptide 101 is presented by the 3502 allele and recognized by HD4-derived T cells (Fig. 3j), whereas peptide 101 is expressed by the HLA-B * 3501 and recognized by HD5-derived T cells ( FIG. 3 k ); peptide VLDFAPPGA (SEQ ID NO: 157) is presented by HLA-A * We showed that the antibody was presented by .0201 and recognized by HD10-derived T cells (FIG. 3l).

[0349] The sequences of WT1 peptides recognized by WT1-specific T cells amplified from HD1-HD10 are shown in Table 3 below.

[0350] [Table 3] TIFF2025072432000032.tif104168

[0351] [Example 3] WT1-specific T cells selectively eliminate WT1-expressing cells. To determine the HLA restriction of WT1-specific T cells and their ability to eliminate WT1-expressing cells, T cells were cocultured with different target cells.

[0352] HD1-derived T cells HLA-A *Considering HD1 carrying the 0201 allele, we tested enriched WT1-specific T cells against different target cells: T2 cells pulsed with overlapping peptide pools containing peptides 40 and 41 (see Table 3); T2 cells pulsed with MelanA / MART1 pool as a negative control (T2 MelanA / MART1 pool); or HLA-A * K562 cells genetically modified to express the 0201 allele and overexpress the WT1 protein (K562 HLA-A * 0201 WT1).

[0353] After 6 hours of co-culture, the expression of CD107a was established by FACS. Results for HD1 showed that after co-culture with T2 cells pulsed with the WT1 pool, the expression of CD8 + The expression of CD107a in >60% of T cells was observed (Figure 4a). Similarly, the expression of CD107a in genetically modified K562 cells (HLA-A * Co-culture with 0201 allele and WT1 protein increased CD8 + This resulted in CD107a expression by >60% of T cells (Figure 4a).

[0354] In contrast, CD8 + CD107a expression by T cells was minimal (Figure 4a).

[0355] These results suggest that isolated, HD1-derived T cells express HLA-A * The results show that when presented by MHC molecules encoded by the 0201 allele, HD1-derived T cells specifically recognize a WT1 peptide containing the sequence APVLDFAPPGA (SEQ ID NO: 117). Furthermore, the results show that HD1-derived T cells can specifically target cells overexpressing the WT1 protein. Thus, these experimental data demonstrate that the TCR expressed by HD1-derived T cells specifically binds to a peptide containing the APVLDFAPPGA (SEQ ID NO: 117) amino acid sequence, and that such a TCR ... *0201 indicates binding.

[0356] HD3-derived T cells HLA-A * Considering HD3 carrying the 0201 allele, we investigated the enriched WT1-specific T cells by immunizing them with different target cells: T2 cells pulsed with subpool 16 (T2-SP16), previously determined to contain immunogenic peptides that elicit an immune response; T2 cells pulsed with the MelanA / MART1 pool as a negative control (T2-MelanA); wild-type K562 cells (K562) as a negative control; or HLA-A * They were co-cultured with K562 cells genetically modified to express the 0201 allele and to overexpress the WT1 protein (K562 A2+WT1+).

[0357] After 4 days of coculture, the ability of HD3-derived T cells to kill target cells was expressed as a shedding index, calculated as the total number of target cells still present after coculture with WT1-specific T cells divided by the total number of target cells alone.

[0358] The results demonstrate the ability of WT1-specific T cells to eliminate target cells expressing the identified specific WT1 epitopes (FIG. 4b). In particular, HD3-derived T cells eliminated approximately 95% of T2 cells pulsed with a WT1 peptide containing the amino acid sequence APVLDFAPPGA (SEQ ID NO: 117) (subpool 16; SP16). Furthermore ... * Approximately 78% of K562 cells expressing MHC molecules encoded by the 0201 allele and overexpressing the WT1 protein were eliminated. In contrast, none of the T2 cells pulsed with the negative control MelanA / MART1 pool were eliminated by HD3-derived T cells. Similarly, elimination of control wild-type K562 cells was minimal (Figure 4b).

[0359] These results demonstrate that isolated, HD3-derived T cells specifically recognize the WT1 peptide containing the amino acid sequence APVLDFAPPGA (SEQ ID NO: 117). Furthermore, the results demonstrate that HD3-derived T cells specifically recognize the WT1 peptide containing the amino acid sequence APVLDFAPPGA (SEQ ID NO: 117). * These experimental data therefore demonstrate that TCRs expressed by HD3-derived T cells can specifically target and kill cells overexpressing the WT1 protein through peptide presentation by MHC encoded by HLA-A 0201. * 0201 Demonstrate that it is restrictive.

[0360] HD4-derived T cells The ability of HD4-derived T cells to eliminate target cells was evaluated by screening T cells for high expression of WT1 antigen and HLA typing (HLA-B * Primary leukemic blasts (CD33 3502) isolated from acute myeloid leukemia (AML) patients selected based on + As a negative control, HD4-derived T cells were co-cultured with HLA-B * They were co-cultured with leukemic blasts from an AML patient that did not express the 3502 allele.

[0361] After 3 days of co-culture at an effector-to-target ratio of 10:1, FACS analysis revealed that HD4 WT1-specific T cells (CD3 + After co-culture with IgG4+ / IgG4+ cells, HLA-B * Leukemic blast cells (CD33 + ) (Figure 4c, upper panel). Indeed, only 0.54% of the remaining total cell population was positive for CD33 expression.

[0362] In contrast, after coculture of WT1-specific T cells with unrelated control AML blasts, CD33 +No cell clearance was observed (FIG. 4c, lower panel). Indeed, in control samples, 7.9% of the total cell population was positive for CD33 expression after coculture with HD4-derived WT1-specific T cells.

[0363] Importantly, these results suggest that HD4-derived WT1-specific T cells express WT1 and HLA-B * This demonstrates the ability to specifically target and kill leukemic blasts (AML cancer cells) that overexpress the MHC encoded by HD4. Thus, HD4-derived TCRs bind to HLA-B * Cancer cells can be specifically targeted and killed in a 3502-restricted manner.

[0364] [Example 4] Immune profiling of Vβ sequences in WT1-specific T cells To better identify the TCRs involved in antigen recognition by WT1-specific T cells from HD1-6 and 10 (it was not possible to perform Vβ immune profiling analysis in HD7, HD8 and HD9 due to reduced cell fitness), we first performed multiparameter FACS analysis to quantitatively determine the TCR Vβ repertoire. Therefore, to determine the clonality of the expanded WT1-specific T cells, we used the IO Test Betamark TCR Vβ Repertoire Kit, following the manufacturer's recommendations. This kit allows the detection of the expression of 24 different Vβ genes in 8 individual tubes. Notably, a coverage of 75% of the complete repertoire of Vβ is guaranteed by using this approach. The results of FACS staining showed a high prevalence of specific Vβ for HD1, HD2, HD3, HD5 - see Figure 5. In HD4, HD6 and HD10, it was not possible to comprehensively determine the dominant Vβ, probably due to the intrinsic limitation of the kit, which contains antibodies covering 75% of the Vβ proteins present.

[0365] [Example 5] High-throughput sequencing of TCR α and β chains isolated from WT1-specific T cells. WT1-specific T cells were harvested at different time points over the co-culture time frame, and their RNA was extracted by using the Arcturus Pico Pure RNA Extraction Kit. After cDNA synthesis, a modified RACE approach was used to amplify the CDR3 sequences of WT1-specific T cells, including magnetic capture to increase the specificity of the reaction and remove unwanted templates (Ruggiero et al., Nat. Commun., 6, 8081, (2015)). Samples were sequenced using an Illumina MiSeq sequencer, and CDR3 clonotypes were identified using MiXTCR software (Bolotin, DA et al., Nature Methods, 12, 380-381, (2015)). In addition, CDR1, CDR2 and CDR3 were further determined using the IMGT V-quest tool (Brochet, X. et al., Nucl. Acids Res., 36, W503-508, (2008), PMID: 18503082; Giudicelli, V., Brochet, X., Lefranc, M.-P., Cold Spring Harb Protoc., June 1, 2011; 2011(6), pii: pdb.prot5633. doi: 10.1101 / pdb.prot5633, PMID: 21632778, abstract also available in the IMGT booklet provided by Cold Spring Harbor (CSH) Protocol).

[0366] Sequencing results showed that for both TCR chains in HD1-10, specific CDR3 clonotypes became dominant over time in the WT1-specific T cell population. The dominant α and β chain genotypes and CDR3 sequences are provided in FIG.

[0367] In addition, the full length α and β chain amino acid sequences for the TCR from HD1-10 were determined - see Table 1, and the corresponding nucleotide sequences were determined - see Table 2.

[0368] [Example 6 Verification of the function of newly identified TCR] HLA-A isolated from HD1 and HD3* TCR α and β sequences that recognize the WT1 VLDFAPPGA (SEQ ID NO: 157) peptide when presented by the 0201 allele were cloned into a lentiviral vector under the control of a bidirectional promoter to drive robust and synchronous expression of both TCR chains in transduced lymphocytes. T cells from healthy individuals were transduced with viral vectors encoding either the HD1 TCR or the HD3 TCR. As a control, we also transduced cells with the WT1 126-134 TCR. Transduced T cells were then transduced with T2 cells (Figure 7a), pulsed with one of the two recognized peptides (VLDFAPPGA (SEQ ID NO: 157) for HD1 and HD3 TCRs; RMFPNAPYL (SEQ ID NO: 255) for WT1 126-134 TCR), wild type or HLA-A * K562 cells derived from three AML patients and either engineered to express the 0201 allele (Fig. 7b) * Functional validation was performed by co-culture with different target cells represented by primary AML blasts (Figure 7c) selected according to their expression of the 0201 allele and WT1 expression. During 3 days of co-culture, we observed that HLA-A * We observed the capacity of each transduced T cell population in specific recognition of the target peptide when presented by the 0201 allele (Figure 7a) and the higher capacity of HD1 T cells in mediating almost complete elimination of engineered K562 cells. The higher capacity of HD1 TCR in recognizing the target antigen was further confirmed by the results of co-culture with pAML blasts. Thus, we demonstrated that the HLA-A mediated IL-1 ... * Higher clearance of both pAML blasts carrying the 0201 allele was observed.

[0369] Materials and Methods The WT1 protein sequence previously published by Gessler et al. (Gessler, M et al., (1990), Nature, 343:774-778) was used to design peptides used for stimulating and isolating WT1-specific T cells. This sequence contains 575 amino acids, including the first 126 amino acids at the N-terminus that are missing in the (exon 5+, KTS+) isoform of WT1. We designed 141 pentadecapeptides spanning the entire sequence of the WT1 protein, each overlapping the next by 11 amino acids.

[0370] Peptides with verified sequences, 70% purity, sterility and absence of endotoxin were synthesized by PRIMM. These peptides were mixed in equal amounts in the WT1 pool at a concentration of 1 μg / ml per peptide. In addition, 24 subpools were generated, each containing up to 12 peptides (4.17 μg / ml per peptide), according to a specific mapping matrix, so that each peptide was only included in two overlapping subpools, as shown in Table 4 (see the mapping grid strategy in Doubrovina, E. et al., Blood, 120, 1633-1646, (2012)).

[0371] [Table 4]

[0372] Isolation of peripheral blood mononuclear cells Peripheral blood was obtained from 10 healthy donors with informed consent at San Raffaele Hospital. Peripheral blood mononuclear cells were isolated using Ficoll-Hypaque density gradient centrifugation.

[0373] immortalized B cells Autologous B cells were isolated from PBMCs of healthy donors using CD19 microbeads (Miltenyi Biotech). Cells were transduced with lentiviral vectors carrying the BCL-6 / BCL-XL transgene (Kwakkenbos, MJ et al., Nat. Med., 16, 123, (2009)) and H / F pseudotype (Levy, C. et al., Molecular Therapy, 20, 9, 1699-1712, (2012)) and cultured in IMDM supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and 10 ng / ml IL21 (Miltenyi Biotech). B cells were restimulated every 5 days by co-culture with irradiated (50 Gy) mouse L cell fibroblasts expressing CD40L (3T3-CD40L) at a B cell:3T3-CD40L ratio of 10:1.

[0374] cell line The present inventors cultured T2 and K562 cell lines in RPMI1640 (GIBCO-BRL) supplemented with penicillin, streptomycin, glutamine and 10% FBS (BioWhittaker).

[0375] leukemia cells Primary AML cells were obtained from the San Raffaele Hospital (OSR) Leukemia Biobank and selected according to WT1 expression and HLA typing by quantitative PCR. All EBV-BLCL and primary leukemia cells were typed for HLA-A, HLA-B, HLA-C, HLA-DR and HLA-DQ alleles at high resolution in the HLA laboratory of the OSR.

[0376] Flow cytometry We used FITC-, PE-, PerCP-, APC-, PE-Cychrome 7-, APC-Cychrome 7-, Pacific Blue and Brilliant Violet conjugated antibodies against human CD3, CD4, CD8, CD107a, IFNγ, TNFα, CD33, CD117, CD34, CD14, Vβ21.3, Vβ8, Vβ7.2 and HLA-A2. APC fluorescently labeled WT1 VLDFAPPGA (SEQ ID NO: 157) and PE fluorescently labeled WT1 RMFPNAPYL (SEQ ID NO: 255) dextramers were used according to the manufacturer's instructions. Cells were incubated with the antibodies for 15 minutes at 4°C and washed with phosphate buffered saline containing 1% FBS. Samples were run on a fluorescence activated cell sorter (FACS) Canto II flow cytometer (BD Biosciences) and data were analyzed by Flow Jo software (Tree star Inc). For intracellular assessment of cytokine secretion and expression of degranulation markers, the Fix / Perm buffer set (Biolegend) was used according to the manufacturer's instructions.

[0377] Stimulation, isolation and expansion of WT1-specific T cells Freshly isolated PBMCs were resuspended in X-VIVO supplemented with 5% human AB serum, 2 mM glutamine and 1 μg / ml CD28 monoclonal antibody and incubated for 10 7 Cells were seeded at a density of 100 cells / ml and stimulated with a WT1 overlapping peptide pool, with each peptide present at a concentration of 1 μg / ml.

[0378] Antigen-specific T cells were isolated after 26–30 h by CD137 expression. More specifically, cells were stained with PE-conjugated CD137 antibody and sorted using anti-PE microbeads (Miltenyi Biotech). - From the fraction, CD3 cells were depleted using CD3-microbeads (Miltenyi Biotech) and irradiated with 30 Gy, with a ratio of 100:1 where possible or at least 20:1, and 5 × 10 6 CD137 at a final density of 10 cells / ml +The peptide-loaded APCs were used in co-culture with the fractions. X-VIVO supplemented with 5% human AB serum, 5ng / ml IL7, 5ng / ml IL15, and 10ng / ml IL21 was used as the medium. The medium containing cytokines was replaced every 2-3 days.

[0379] Restimulation of expanded antigen-specific T cells Cells were restimulated every 7-14 days with autologous APCs pulsed with WT1 (PBMC CD3-depleted cells; immortalized B cells). For the first restimulation, cells were washed 2 days prior and seeded in cytokine-free medium. APCs were irradiated with 30 Gy, pulsed overnight with peptide pools, and co-cultured with effector cells in X-VIVO supplemented with 5% human AB serum, 1 μg / ml CD28 monoclonal antibody, and IL7 (5 ng / ml), IL15 (5 ng / ml), IL21 (10 ng / ml).

[0380] Assessment of T cell responses The percentage of T cells responding to WT1 peptide pools was measured by performing 6-hour co-cultures (at least 1:1 ratio) of effector cells with autologous APCs pulsed with the desired antigen (WT1 peptide pool, WT1 subpools, WT1 individual peptides, irrelevant peptide pool as control). Co-cultures were seeded in X-VIVO supplemented with 5% human AB serum and, for evaluation of degranulation, CD28 monoclonal antibody (1 μg / ml), Golgi Stop (BD) and CD107a-FITC antibody. Cells were then fixed, permeabilized and intracellularly stained to identify CD3 T cells expressing IFNγ and CD107a. + CD8 + or CD3 + CD4 + The percentage of cells was determined.

[0381] Immunogenic peptide mapping T cells stimulated with the WT1 pool were seeded in different wells and co-cultured with autologous APCs loaded with each one of the WT1 subpools at a ratio of at least 1:1. T cell responses to each subpool were measured by FACS analysis as previously described. Deconvolution of the mapping grid was essential to determine which shared peptides were eliciting T cell responses. Once the immunogenic peptides were determined, T cells were further stimulated with APCs loaded with the individual peptides to confirm their immunogenicity.

[0382] Assessment of the ability of T cells to recognize WT1-expressing cells The WT1 specificity and HLA restriction ability of T cells to recognize target cells were measured using different experimental methods. For HD1-derived T cells, CD107a secretion was determined by FACS analysis after 6 hours of co-culture with target cells, for HD3-derived T cells, the shedding index was calculated as the total number of target cells present after 4 days of co-culture with WT1-specific T cells divided by the total number of target cells alone, and for HD4-derived T cells, the CD3 + CD33 still present after 3 days of co-culture with WT1-specific T cells + The percentage of target cells (AML primary cells carrying the HLA allele of interest and, as a control, AML primary cells not carrying the specific HLA allele) was assessed by cytofluorometry analysis.

[0383] Assessment of T cell clonality To determine the clonality of the expanded WT1-specific T cells, the IO Test Betamark TCR V beta repertoire kit was used according to the manufacturer's recommendations.

[0384] TCR repertoire sequencing WT1-specific T cells were harvested at different time points over the co-culture time frame, and RNA was extracted by using the Arcturus Pico Pure RNA extraction kit. Complementarity determining region (CDR) 3 sequences of WT1-specific T cells were amplified by using a modified RACE approach (Ruggiero, E. et al., Nat. Commun, 6, 8081, (2015)). Samples were sequenced by using an Illumina MiSeq sequencer, and CDR3 clonotypes were identified using MiXCR software (Bolotin, DA et al., Nature Methods, 12, 380-381, (2015)).

[0385] Lentiviral Vectors The TCR α and β chain genes isolated from HD1 and HD3 were codon-optimized, cysteine-engineered, and cloned into a lentiviral vector (LV) under a bidirectional promoter. The amino acid (aa) and nucleotide (nt) sequences are as follows:

[0386] [Table 5] TIFF2025072432000035.tif250162TIFF2025072432000036.tif210163

[0387] LVs were packaged with integrase-competent third generation constructs and pseudotyped with the vesicular stomatitis virus (VSV) envelope. As a control, we included LVs encoding the WT1 126-134 TCR, which recognizes the RMFPNAPYL (SEQ ID NO: 255) peptide.

[0388] Vector transduction For transduction with WT1-TCR lentiviral vectors, T lymphocytes isolated from healthy individuals were activated and sorted using magnetic beads coupled to antibodies against CD3 and CD28 (ClinExVivo CD3 / CD28; Invitrogen) according to the manufacturer's instructions, and cultured in Iscove's modified Dulbecco's medium (IMDM) (GIBCO-BRL) supplemented with penicillin, streptomycin, 10% FBS, and 5 ng / ml each of IL-7 and IL-15 (PeproTech). For transduction, T lymphocytes were cultured at 2.5 × 10 6 T cells were then seeded at 10 6 The cells were cultured and expanded at 1000 cells / ml. Transduction efficiency was determined by measuring the percentage of CD3 T cells expressing specific dextramers. Cells were transduced with APC or PE fluorescently labeled HLA-A specific for VLDFAPPGA (SEQ ID NO: 157) or RMFPNAPYL (SEQ ID NO: 255) peptides using anti-APC or anti-PE microbeads (Miltenyi Biotec) according to the manufacturer's instructions. * Fractionation was performed using 0201 Dextramer (Immudex).

[0389] Functional assays The ability of T cells transfected with HD1, HD3 and WT1 126-134 TCRs to recognize WT1-expressing target cells was examined using (a) T2 cells pulsed with either the WT1 126-134 peptide or the VLDFAPPGA (SEQ ID NO: 157) peptide (effector:target ratio=1:1); (b) wild-type (K562) or HLA-A * (c) K562 cells (effector:target ratio = 1:1) either genetically modified to express the 0201 allele; *The detachment index was measured upon co-culture with three different primary AML blasts (effector:target ratio=5:1), selected according to the expression of the 0201 allele and of the WT1 antigen. For co-culture with T2 and K562 cell lines, we included non-transduced T cells as a control. After 3 days of culture, the percentage of target cells was assessed by cytofluorimetric analysis. The detachment index was calculated as follows: 1-(total number of target cells still present after 3 days of co-culture with WT1-specific T cells / total number of target cells alone).

[0390] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the invention described will be apparent to those of skill in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods of practicing the invention that are obvious to those skilled in the art of cell biology, immunology, immunotherapy, molecular biology, oncology, or related fields are intended to be within the scope of the appended claims.

Claims

1. 1. A T cell receptor (TCR) that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by a major histocompatibility complex (MHC), comprising: (i) a CDR3α comprising the amino acid sequence of CGTAWINDYKLSF (SEQ ID NO: 3), or a variant thereof having up to three amino acid substitutions, additions or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRKTGGYSNQPQHF (SEQ ID NO: 8), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (ii) a CDR3α comprising the amino acid sequence of CVVNLLSNQGGKLIF (SEQ ID NO: 36), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSQDYLVSNEKLFF (SEQ ID NO: 41), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (iii) a CDR3α comprising the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (iv) a CDR3α comprising the amino acid sequence of CAVRLSGSARQLTF (SEQ ID NO: 14), or a variant thereof having up to three amino acid substitutions, additions or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO:30), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (v) a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLLGDEQYF (SEQ ID NO: 24), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (vi) a CDR3α comprising the amino acid sequence of CAYRSLKYGNKLVF (SEQ ID NO: 19), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLVALQGAGEQYF (SEQ ID NO:30), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (vii) a CDR3α comprising the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO:57), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (viii) a CDR3α comprising the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASTQTPYEQYF (SEQ ID NO:63), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (ix) a CDR3α comprising the amino acid sequence of CATDAYSGNTPLVF (SEQ ID NO: 47), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSTVGGEDYGYTF (SEQ ID NO:69), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (x) a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRAAGLDTEAFF (SEQ ID NO:57), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xi) a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASTQTPYEQYF (SEQ ID NO:63), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xii) a CDR3α comprising the amino acid sequence of CAVRAEIYNQGGKLIF (SEQ ID NO: 52), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSTVGGEDYGYTF (SEQ ID NO:69), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xiii) a CDR3α comprising the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CAISVGQGALYEQYF (SEQ ID NO: 80), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xiv) a CDR3α comprising the amino acid sequence of CAASMAGAGSYQLTF (SEQ ID NO: 75), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSVARDRRNYGYTF (SEQ ID NO:86), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xv) a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSDTRAREQFF (SEQ ID NO:97), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xvi) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xvii) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xviii) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xix) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xx) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxi) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxii) a CDR3α comprising the amino acid sequence of CAVTVGNKLVF (SEQ ID NO: 175), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASRGWREQFF (SEQ ID NO: 180), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxiii) a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSWGYQETQYF (SEQ ID NO: 196), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxiv) a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxv) a CDR3α comprising the amino acid sequence of CAARSYNTDKLIF (SEQ ID NO: 186), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxvi) a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSWGYQETQYF (SEQ ID NO: 196), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxvii) a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPTGGEYYGYTF (SEQ ID NO: 202), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxviii) a CDR3α comprising the amino acid sequence of CAASYNNARLMF (SEQ ID NO: 191), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxix) a CDR3α comprising the amino acid sequence of CAASGGRDDKIIF (SEQ ID NO: 214), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSYSRTESTDTQYF (SEQ ID NO: 219), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxx) a CDR3α comprising the amino acid sequence of CAANNARLMF (SEQ ID NO: 92), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxi) a CDR3α comprising the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSDTRAREQFF (SEQ ID NO:97), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxii) a CDR3α comprising the amino acid sequence of CAASATGNQFYF (SEQ ID NO: 266), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSPGQHGELFF (SEQ ID NO: 271), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxiii) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDSVSGNTIYF (SEQ ID NO: 163), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxiv) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSVGGSGSYNEQFF (SEQ ID NO: 169), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxv) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxvi) a CDR3α comprising the amino acid sequence of CATDGDSSYKLIF (SEQ ID NO: 277), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxvii) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxviii) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxix) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CASSLGLSISQETQYF (SEQ ID NO: 288), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxx) a CDR3α comprising the amino acid sequence of CAERLNTDKLIF (SEQ ID NO: 103), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions, or deletions; (xxxxi) a CDR3α comprising the amino acid sequence of CAVEATDSWGKLQF (SEQ ID NO: 108), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions; or (xxxxii) a CDR3α comprising the amino acid sequence of CAVRTSYDKVIF (SEQ ID NO: 113), or a variant thereof having up to three amino acid substitutions, additions, or deletions; and It comprises a CDR3β comprising the amino acid sequence of CSARDVLTGDYGYTF (SEQ ID NO: 282), or a variant thereof having up to three amino acid substitutions, additions or deletions.

2. The TCR of claim 1, comprising the following CDR sequences: (i) CDR1α - KALYS (SEQ ID NO: 1), CDR2α - LLKGGEQ (SEQ ID NO: 2), CDR3α - CGTAWINDYKLSF (SEQ ID NO: 3), CDR1β - SGHDY (SEQ ID NO: 6), CDR2β - FNNNVP (SEQ ID NO: 7), and CDR3β - CASRKTGGYSNQPQHF (SEQ ID NO: 8), or each of these variants having up to three amino acid substitutions, additions or deletions; (ii) CDR1α - NSASQS (SEQ ID NO: 34), CDR2α - VYSSGN (SEQ ID NO: 35), CDR3α - CVVNLLSNQGGKLIF (SEQ ID NO: 36), CDR1β - LGHNA (SEQ ID NO: 39), CDR2β - YSLEER (SEQ ID NO: 40), and CDR3β - CASSQDYLVSNEKLFF (SEQ ID NO: 41), or each of these variants having up to three amino acid substitutions, additions or deletions; (iii) CDR1α -SSVPPY (SEQ ID NO: 12), CDR2α - YTSAATLV (SEQ ID NO: 13), CDR3α - CAVRLSGSARQLTF (SEQ ID NO: 14), CDR1β - SGHAT (SEQ ID NO: 22), CDR2β - FQNNGV (SEQ ID NO: 23), and CDR3β - CASSLLGDEQYF (SEQ ID NO: 24), or each of these variants having up to three amino acid substitutions, additions or deletions; (iv) CDR1α -SSVPPY (SEQ ID NO: 12), CDR2α - YTSAATLV (SEQ ID NO: 13), CDR3α - CAVRLSGSARQLTF (SEQ ID NO: 14), CDR1β - SGHTA (SEQ ID NO: 28), CDR2β - FQGNSA (SEQ ID NO: 29), and CDR3β - CASSLVALQGAGEQYF (SEQ ID NO: 30), or each of these variants having up to three amino acid substitutions, additions or deletions; (v) CDR1α - TSESDYY (SEQ ID NO: 17), CDR2α - QEAYKQQN (SEQ ID NO: 18), CDR3α - CAYRSLKYGNKLVF (SEQ ID NO: 19), CDR1β - SGHAT (SEQ ID NO: 22), CDR2β - FQNNGV (SEQ ID NO: 23), and CDR3β - CASSLLGDEQYF (SEQ ID NO: 24), or each of these variants having up to three amino acid substitutions, additions or deletions; (vi) CDR1α - TSESDYY (SEQ ID NO: 17), CDR2α - QEAYKQQN (SEQ ID NO: 18), CDR3α - CAYRSLKYGNKLVF (SEQ ID NO: 19), CDR1β - SGHTA (SEQ ID NO: 28), CDR2β - FQGNSA (SEQ ID NO: 29), and CDR3β - CASSLVALQGAGEQYF (SEQ ID NO: 30), or each of these variants having up to three amino acid substitutions, additions or deletions; (vii) CDR1α - TSINN (SEQ ID NO: 45), CDR2α - IRSNERE (SEQ ID NO: 46), CDR3α - CATDAYSGNTPLVF (SEQ ID NO: 47), CDR1β - MNHNS (SEQ ID NO:55), CDR2β - SASEGT (SEQ ID NO:56), and CDR3β - CASRAAGLDTEAFF (SEQ ID NO:57), or each of these variants having up to three amino acid substitutions, additions or deletions; (viii) CDR1α - TSINN (SEQ ID NO: 45), CDR2α - IRSNERE (SEQ ID NO: 46), CDR3α - CATDAYSGNTPLVF (SEQ ID NO: 47), CDR1β - MNHNY (SEQ ID NO: 61), CDR2β - SVGAGI (SEQ ID NO: 62), and CDR3β - CASTQTPYEQYF (SEQ ID NO: 63), or each of these variants having up to three amino acid substitutions, additions or deletions; (ix) CDR1α - TSINN (SEQ ID NO: 45), CDR2α - IRSNERE (SEQ ID NO: 46), CDR3α - CATDAYSGNTPLVF (SEQ ID NO: 47), CDR1β - SGHNS (SEQ ID NO: 67), CDR2β - FNNNVP (SEQ ID NO: 68), and CDR3β - CASSTVGGEDYGYTF (SEQ ID NO: 69), or each of these variants having up to three amino acid substitutions, additions or deletions; (x) CDR1α - DSAIYN (SEQ ID NO: 50), CDR2α - IQSSQRE (SEQ ID NO:51), CDR3α - CAVRAEIYNQGGKLIF (SEQ ID NO:52), CDR1β - MNHNS (SEQ ID NO:55), CDR2β - SASEGT (SEQ ID NO:56), and CDR3β - CASRAAGLDTEAFF (SEQ ID NO:57), or each of these variants having up to three amino acid substitutions, additions or deletions; (xi) CDR1α - DSAIYN (SEQ ID NO: 50), CDR2α - IQSSQRE (SEQ ID NO:51), CDR3α - CAVRAEIYNQGGKLIF (SEQ ID NO:52), CDR1β - MNHNY (SEQ ID NO: 61), CDR2β - SVGAGI (SEQ ID NO: 62), and CDR3β - CASTQTPYEQYF (SEQ ID NO: 63), or each of these variants having up to three amino acid substitutions, additions or deletions; (xii) CDR1α - DSAIYN (SEQ ID NO: 50), CDR2α - IQSSQRE (SEQ ID NO:51), CDR3α - CAVRAEIYNQGGKLIF (SEQ ID NO:52), CDR1β - SGHNS (SEQ ID NO: 67), CDR2β - FNNNVP (SEQ ID NO: 68), and CDR3β - CASSTVGGEDYGYTF (SEQ ID NO: 69), or each of these variants having up to three amino acid substitutions, additions or deletions; (xiii) CDR1α - DSASNY (SEQ ID NO: 73), CDR2α - IRSNVGE (SEQ ID NO: 74), CDR3α - CAASMAGAGSYQLTF (SEQ ID NO: 75), CDR1β - ENHRY (SEQ ID NO:78), CDR2β - SYGVKD (SEQ ID NO: 79), and CDR3β - CAISVGQGALYEQYF (SEQ ID NO: 80), or each of these variants having up to three amino acid substitutions, additions or deletions; (xiv) CDR1α - DSASNY (SEQ ID NO: 73), CDR2α - IRSNVGE (SEQ ID NO: 74), CDR3α - CAASMAGAGSYQLTF (SEQ ID NO: 75), CDR1β - SGDLS (SEQ ID NO: 84), CDR2β - YYNGEE (SEQ ID NO: 85), and CDR3β - CASSVARDRRNYGYTF (SEQ ID NO: 86), or each of these variants having up to three amino acid substitutions, additions or deletions; (xv) CDR1α - NSMFDY (SEQ ID NO: 90), CDR2α - ISSIKDK (SEQ ID NO: 91), CDR3α - CAANNARLMF (SEQ ID NO: 92), CDR1β - SGHNS (SEQ ID NO: 95), CDR2β - FNNNVP (SEQ ID NO: 96), and CDR3β - CASSDTRAREQFF (SEQ ID NO: 97), or each of these variants having up to three amino acid substitutions, additions or deletions; (xvi) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xvii) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xviii) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xix) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xx) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxi) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxii) CDR1α - VGISA (SEQ ID NO: 173), CDR2α - LSSGK (SEQ ID NO: 174), CDR3α - CAVTVGNKLVF (SEQ ID NO: 175), CDR1β - MNHNS (SEQ ID NO: 178), CDR2β - SASEGT (SEQ ID NO: 179), and CDR3β - CASRGWREQFF (SEQ ID NO: 180), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxiii) CDR1α - VGISA (SEQ ID NO: 184), CDR2α - LSSGK (SEQ ID NO: 185), CDR3α - CAARSYNTDKLIF (SEQ ID NO: 186), CDR1β - SGHTS (SEQ ID NO: 194), CDR2β - YDEGEE (SEQ ID NO: 195), and CDR3β - CASSWGYQETQYF (SEQ ID NO: 196), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxiv) CDR1α - VGISA (SEQ ID NO: 184), CDR2α - LSSGK (SEQ ID NO: 185), CDR3α - CAARSYNTDKLIF (SEQ ID NO: 186), CDR1β - KGHSH (SEQ ID NO: 200), CDR2β - LQKENI (SEQ ID NO: 201), and CDR3β - CASSPTGGEYYGYTF (SEQ ID NO: 202), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxv) CDR1α - VGISA (SEQ ID NO: 184), CDR2α - LSSGK (SEQ ID NO: 185), CDR3α - CAARSYNTDKLIF (SEQ ID NO: 186), CDR1β - MNHEY (SEQ ID NO: 206), CDR2β - SVGAGI (SEQ ID NO: 207), and CDR3β - CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxvi) CDR1α - NSMFDY (SEQ ID NO: 189), CDR2α - ISSIKDK (SEQ ID NO: 190), CDR3α - CAASYNNARLMF (SEQ ID NO: 191), CDR1β - SGHTS (SEQ ID NO: 194), CDR2β - YDEGEE (SEQ ID NO: 195), and CDR3β - CASSWGYQETQYF (SEQ ID NO: 196), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxvii) CDR1α - NSMFDY (SEQ ID NO: 189), CDR2α - ISSIKDK (SEQ ID NO: 190), CDR3α - CAASYNNARLMF (SEQ ID NO: 191), CDR1β - KGHSH (SEQ ID NO: 200), CDR2β - LQKENI (SEQ ID NO: 201), and CDR3β - CASSPTGGEYYGYTF (SEQ ID NO: 202), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxviii) CDR1α - NSMFDY (SEQ ID NO: 189), CDR2α - ISSIKDK (SEQ ID NO: 190), CDR3α - CAASYNNARLMF (SEQ ID NO: 191), CDR1β - MNHEY (SEQ ID NO: 206), CDR2β - SVGAGI (SEQ ID NO: 207), and CDR3β - CASSSYPLRTGRYNSYNSPLHF (SEQ ID NO: 208), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxix) CDR1α - NSMFDY (SEQ ID NO: 212), CDR2α - ISSIKDK (SEQ ID NO: 213), CDR3α - CAASGGRDDKIIF (SEQ ID NO: 214), CDR1β - MNHEY (SEQ ID NO: 217), CDR2β - SVGAGI (SEQ ID NO: 218), and CDR3β - CASSYSRTESTDTQYF (SEQ ID NO: 219), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxx) CDR1α - NSMFDY (SEQ ID NO: 90), CDR2α - ISSIKDK (SEQ ID NO: 91), CDR3α - CAANNARLMF (SEQ ID NO: 92), CDR1β - SGHRS (SEQ ID NO: 269), CDR2β - YFSETQ (SEQ ID NO: 270), and CDR3β - CASSPGQHGELFF (SEQ ID NO: 271), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxi) CDR1α - NSMFDY (SEQ ID NO: 264), CDR2α - ISSIKDK (SEQ ID NO: 265), CDR3α - CAASATGNQFYF (SEQ ID NO: 266), CDR1β - SGHNS (SEQ ID NO: 95), CDR2β - FNNNVP (SEQ ID NO: 96), and CDR3β - CASSDTRAREQFF (SEQ ID NO: 97), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxii) CDR1α - NSMFDY (SEQ ID NO: 264), CDR2α - ISSIKDK (SEQ ID NO: 265), CDR3α - CAASATGNQFYF (SEQ ID NO: 266), CDR1β - SGHRS (SEQ ID NO: 269), CDR2β - YFSETQ (SEQ ID NO: 270), and CDR3β - CASSPGQHGELFF (SEQ ID NO: 271), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxiii) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - DFQATT (SEQ ID NO: 161), CDR2β - SNEGSKA (SEQ ID NO: 162), and CDR3β - CSARDSVSGNTIYF (SEQ ID NO: 163), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxiv) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - SQVTM (SEQ ID NO: 167), CDR2β - ANQGSEA (SEQ ID NO: 168), and CDR3β - CSVGGSGSYNEQFF (SEQ ID NO: 169), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxv) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxvi) CDR1α - TSINN (SEQ ID NO: 275), CDR2α - IRSNERE (SEQ ID NO: 276), CDR3α - CATDGDSSYKLIF (SEQ ID NO: 277), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxvii) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxviii) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxix) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - SGHDY (SEQ ID NO: 286), CDR2β - FNNNVP (SEQ ID NO: 287), and CDR3β - CASSLGLSISQETQYF (SEQ ID NO: 288), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxx) CDR1α - DSSSTY (SEQ ID NO: 101), CDR2α - IFSNMDM (SEQ ID NO: 102), CDR3α - CAERLNTDKLIF (SEQ ID NO: 103), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions; (xxxxi) CDR1α - DSVNN (SEQ ID NO: 106), CDR2α - IPSGT (SEQ ID NO: 107), CDR3α - CAVEATDSWGKLQF (SEQ ID NO: 108), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions; or (xxxxii) CDR1α - DSASNY (SEQ ID NO: 111), CDR2α - IRSNVGE (SEQ ID NO: 112), CDR3α - CAVRTSYDKVIF (SEQ ID NO: 113), CDR1β - DFQATT (SEQ ID NO: 280), CDR2β - SNEGSKA (SEQ ID NO: 281), and CDR3β - CSARDVLTGDYGYTF (SEQ ID NO: 282), or each of these variants having up to three amino acid substitutions, additions or deletions.

3. The TCR of claim 1 or 2, comprising: (i) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 75% sequence identity thereto; (ii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 75% sequence identity thereto; (iii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least 75% sequence identity thereto; (iv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 75% sequence identity thereto; (v) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least 75% sequence identity thereto; (vi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 75% sequence identity thereto; (vii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least 75% sequence identity thereto; (viii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof having at least 75% sequence identity thereto; (ix) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof having at least 75% sequence identity thereto; (x) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least 75% sequence identity thereto; (xi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof having at least 75% sequence identity thereto; (xii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof having at least 75% sequence identity thereto; (xiii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least 75% sequence identity thereto; (xiv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least 75% sequence identity thereto; (xv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least 75% sequence identity thereto; (xvi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75% sequence identity thereto; (xvii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75% sequence identity thereto; (xviii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75% sequence identity thereto; (xix) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75% sequence identity thereto; (xx) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75% sequence identity thereto; (xxi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75% sequence identity thereto; (xxii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 176, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 181, or a variant thereof having at least 75% sequence identity thereto; (xxiii) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least 75% sequence identity thereto; (xxiv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 203, or a variant thereof having at least 75% sequence identity thereto; (xxv) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 209, or a variant thereof having at least 75% sequence identity thereto; (xxvi) an α chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75% sequence identity thereto; and a β chain variable domain comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least 75% sequence identity thereto; (xxvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 203, or a variant thereof having at least 75% sequence identity thereto; (xxviii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 209, or a variant thereof having at least 75% sequence identity thereto; (xxix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 215, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 220, or a variant thereof having at least 75% sequence identity thereto; (xxx) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 272, or a variant thereof having at least 75% sequence identity thereto; (xxxi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 267, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least 75% sequence identity thereto; (xxxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 267, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 272, or a variant thereof having at least 75% sequence identity thereto; (xxxiii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least 75% sequence identity thereto; (xxxiv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof having at least 75% sequence identity thereto; (xxxv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75% sequence identity thereto; (xxxvi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 278, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75% sequence identity thereto; (xxxvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75% sequence identity thereto; (xxxviii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75% sequence identity thereto; (xxxix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least 75% sequence identity thereto; (xxxx) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75% sequence identity thereto; (xxxxi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75% sequence identity thereto; or (xxxxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 283, or a variant thereof having at least 75% sequence identity thereto.

4. The TCR of any one of claims 1 to 3, comprising: (i) an α chain comprising the amino acid sequence of SEQ ID NO:5, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, and variants of SEQ ID NOs:10 and 11 having at least 75% sequence identity thereto; (ii) an α chain comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, and variants of SEQ ID NOs: 43 and 44 having at least 75% sequence identity thereto; (iii) an α chain comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, and variants of SEQ ID NOs: 26 and 27 having at least 75% sequence identity thereto; (iv) an α chain comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and variants of SEQ ID NOs: 32 and 33 having at least 75% sequence identity thereto; (v) an α chain comprising the amino acid sequence of SEQ ID NO:21, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, and variants of SEQ ID NOs:26 and 27 having at least 75% sequence identity thereto; (vi) an α chain comprising the amino acid sequence of SEQ ID NO:21, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, and variants of SEQ ID NOs:32 and 33 having at least 75% sequence identity thereto; (vii) an α chain comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, and variants of SEQ ID NOs: 59 and 60 having at least 75% sequence identity thereto; (viii) an α chain comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66, and variants of SEQ ID NOs: 65 and 66 having at least 75% sequence identity thereto; (ix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 72, and variants of SEQ ID NOs: 71 and 72 having at least 75% sequence identity thereto; (x) an alpha chain comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, and variants of SEQ ID NOs: 59 and 60 having at least 75% sequence identity thereto; (xi) an α chain comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66, and variants of SEQ ID NOs: 65 and 66 having at least 75% sequence identity thereto; (xii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 72, and variants of SEQ ID NOs: 71 and 72 having at least 75% sequence identity thereto; (xiii) an α chain comprising the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least 75% sequence identity thereto; and a β chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, and variants of SEQ ID NOs: 82 and 83 having at least 75% sequence identity thereto; (xiv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 88, SEQ ID NO: 89, and variants of SEQ ID NOs: 88 and 89 having at least 75% sequence identity thereto; (xv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, and variants of SEQ ID NO: 99 and SEQ ID NO: 100 having at least 75% sequence identity thereto; (xvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75% sequence identity thereto; (xvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75% sequence identity thereto; (xviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75% sequence identity thereto; (xix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75% sequence identity thereto; (xx) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75% sequence identity thereto; (xxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75% sequence identity thereto; (xxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 177, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 182, SEQ ID NO: 183, and variants of SEQ ID NOs: 182 and 183 having at least 75% sequence identity thereto; (xxiii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 198, SEQ ID NO: 199, and variants of SEQ ID NOs: 198 and 199 having at least 75% sequence identity thereto; (xxiv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 204, SEQ ID NO: 205, and variants of SEQ ID NOs: 204 and 205 having at least 75% sequence identity thereto; (xxv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 188, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, and variants of SEQ ID NOs: 210 and 211 having at least 75% sequence identity thereto; (xxvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 198, SEQ ID NO: 199, and variants of SEQ ID NOs: 198 and 199 having at least 75% sequence identity thereto; (xxvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 204, SEQ ID NO: 205, and variants of SEQ ID NOs: 204 and 205 having at least 75% sequence identity thereto; (xxviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, and variants of SEQ ID NOs: 210 and 211 having at least 75% sequence identity thereto; or (xxix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 216, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 221, SEQ ID NO: 222, and variants of SEQ ID NOs: 221 and 222 having at least 75% sequence identity thereto; (xxx) an alpha chain comprising the amino acid sequence of SEQ ID NO:94, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:273, SEQ ID NO:274, and variants of SEQ ID NOs:273 and 274 having at least 75% sequence identity thereto; (xxxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 268, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, and variants of SEQ ID NOs: 99 and 100 having at least 75% sequence identity thereto; (xxxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 268, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 273, SEQ ID NO: 274, and variants of SEQ ID NOs: 273 and 274 having at least 75% sequence identity thereto; (xxxiii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 165, SEQ ID NO: 166, and variants of SEQ ID NOs: 165 and 166 having at least 75% sequence identity thereto; (xxxiv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, and variants of SEQ ID NOs: 171 and 172 having at least 75% sequence identity thereto; (xxxv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75% sequence identity thereto; (xxxvi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 279, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75% sequence identity thereto; (xxxvii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75% sequence identity thereto; (xxxviii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75% sequence identity thereto; (xxxix) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 290, SEQ ID NO: 291, and variants of SEQ ID NOs: 290 and 291 having at least 75% sequence identity thereto; (xxxx) an alpha chain comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75% sequence identity thereto; (xxxxi) an alpha chain comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75% sequence identity thereto; or (xxxxii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least 75% sequence identity thereto; and a beta chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, and variants of SEQ ID NOs: 284 and 285 having at least 75% sequence identity thereto.

5. 23. A T cell receptor (TCR) that binds to a Wilms tumor 1 protein (WT1) peptide when it is presented by the major histocompatibility complex (MHC), wherein the WT1 peptide comprises an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO:123), YESDNHTTPIL (SEQ ID NO:126), NHTTPILCGAQYRIH (SEQ ID NO:127), QCLSAFTVHFSGQFT (SEQ ID NO:118), EDPMGQQGSLGEQQY (SEQ ID NO:119), SQLECMTWNQMNLGA (SEQ ID NO:120), APVLDFAPPGA (SEQ ID NO:117), NQMNLGATLKG (SEQ ID NO:250), DPGGIWAKLGAAEAS (SEQ ID NO:251), NHTTPILCGAQYRIH (SEQ ID NO:252), KRHQRRHTGVKPFQC (SEQ ID NO:253), PSCQKKFARSDELVR (SEQ ID NO:254), and variants of each of these having up to three amino acid substitutions, additions, or deletions.

6. A TCR according to any one of claims 1 to 5, which binds to an MHC I and / or MHC II peptide complex.

7. 7. The TCR of any one of claims 1 to 6, which is restricted to a human leukocyte antigen (HLA) allele, preferably said TCR is restricted to an HLA-A or HLA-B allele, more preferably said TCR is restricted to an HLA-A * 0201, HLA-A * 0101, HLA-A * 2402 and HLA-A * 0301, 0302, or the TCR is restricted to an HLA-A allele selected from the group consisting of HLA-B * 0702, HLA-B * 3501 and HLA-B * 3502.

8. below: HLA-A * (i), (ii), (xv), (xvi), (xxix), (xxx) to (xxxiii), (xxxv) or (xxxx) of any one of claims 1 to 4, which is subject to the provisions of 0201; b. HLA-B * (vii) of any one of claims 1 to 4, which is bound by 3502; or c. HLA-B * 3501, wherein the TCR is any one of (xiii) to (xiv) of any one of claims 1 to 4.

9. A TCR as described in any one of claims 1 to 8, comprising one or more mutations at the α chain / β chain interface such that when the α chain and the β chain are expressed in a T cell, the frequency of mispairing between the chains and the endogenous TCR α and β chains is reduced.

10. The TCR of claim 9, wherein the one or more mutations introduce a cysteine ​​residue into the constant region domain of each of the alpha and beta chains, the cysteine ​​residue being capable of forming a disulfide bond between the alpha and beta chains.

11. A TCR according to any one of claims 1 to 10, comprising a murine constant region.

12. A TCR according to any one of claims 1 to 11, which is a soluble TCR.

13. An isolated polynucleotide encoding the alpha chain of a T cell receptor (TCR) according to any one of claims 1 to 12, and / or the beta chain of a TCR according to any one of claims 1 to 12.

14. 14. The isolated polynucleotide of claim 13, which encodes an alpha chain linked to a beta chain.

15. 15. The isolated polynucleotide of claim 13 or 14, encoding one or more small interfering RNAs (siRNAs) or other agents capable of reducing or suppressing expression of one or more endogenous TCR genes.

16. A vector comprising the polynucleotide of any one of claims 13 to 15.

17. 17. The vector of claim 16, comprising a polynucleotide encoding one or more of the CD3 chains, CD8, a suicide gene, and / or a selection marker.

18. A cell comprising a TCR according to any one of claims 1 to 11, a polynucleotide according to any one of claims 13 to 15, or a vector according to claim 16 or 17, optionally further comprising a vector encoding one or more of the CD3 chains, CD8, a suicide gene, and / or a selection marker.

19. 19. The cell of claim 18, which is a T cell, lymphocyte, or stem cell, optionally wherein the T cell, lymphocyte, or stem cell is selected from the group consisting of CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells, and pluripotent stem cells.

20. The cell of claim 19, which is a T cell isolated from a subject.

21. the endogenous gene encoding the TCR α chain and / or the endogenous gene encoding the TCR β chain is disrupted, preferably so that the endogenous gene encoding the TCR α chain and / or the endogenous gene encoding the TCR β chain is not expressed, Optionally, an endogenous gene encoding a TCR α chain and / or an endogenous gene encoding a TCR β chain is disrupted by insertion of an expression cassette comprising a polynucleotide sequence encoding a TCR according to any one of claims 1 to 11, Optionally, one or more endogenous genes encoding MHC are disrupted, Further optionally, endogenous genes involved in persistence, proliferation, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell functions are disrupted, preferably said endogenous genes involved in persistence, proliferation, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability, or other T cell functions are selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160, and CTLA4. A cell according to any one of claims 18 to 20.

22. A method for the preparation of a cell comprising the step of introducing a vector according to claim 16 and / or 17 into a cell in vitro, ex vivo or in vivo, for example by transfection or transduction.

23. 23. A method for preparing a cell according to claim 22, comprising a step of T cell editing comprising disrupting an endogenous gene encoding a TCR alpha chain and / or an endogenous gene encoding a TCR beta chain with an artificial nuclease, preferably said artificial nuclease being selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

24. A method for preparing the cell of claim 23, comprising a step of targeted integration of an expression cassette into an endogenous gene encoding a TCR alpha chain and / or an endogenous gene encoding a TCR beta chain disrupted by an artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding a TCR according to any one of claims 1 to 11.

25. 25. A method for the preparation of a cell according to any one of claims 22 to 24, comprising a step of disrupting one or more endogenous genes encoding MHC, preferably wherein the cell prepared by said method is a non-allo-reactive universal T cell.

26. 26. A method for preparing a cell according to any one of claims 22 to 25, comprising a step of disrupting one or more endogenous genes so as to modify persistence, proliferation, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability or other T cell function, preferably comprising a step of targeted integration of an expression cassette into an endogenous gene involved in persistence, proliferation, activity, resistance to exhaustion / senescence / inhibitory signals, homing ability or other T cell function, preferably disrupted by an artificial nuclease, said expression cassette comprising a polynucleotide sequence encoding a TCR according to any one of claims 1 to 11, preferably said endogenous gene being selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160 and CTLA4.

27. 27. The cell of any one of claims 18 to 21, or the cell prepared by the method of any one of claims 22 to 26, for use in adoptive cell transfer, preferably adoptive T cell transfer, optionally wherein said adoptive T cell transfer is allogeneic adoptive T cell transfer, autologous adoptive T cell transfer, or universal non-allo-reactive adoptive T cell transfer.

28. A chimeric molecule comprising the TCR of any one of claims 1 to 11, or a part thereof, conjugated to a non-cellular substrate, a toxin and / or an antibody, optionally wherein the non-cellular substrate is selected from the group consisting of nanoparticles, exosomes and other non-cellular substrates.

29. A TCR according to any one of claims 1 to 11, an isolated polynucleotide according to any one of claims 13 to 15, a vector according to claim 16 or 17, a cell according to any one of claims 18 to 21, a cell prepared by the method according to any one of claims 22 to 26, or a chimeric molecule according to claim 28, for use in therapy.

30. A TCR according to any one of claims 1 to 11, an isolated polynucleotide according to any one of claims 13 to 15, a vector according to claim 16 or 17, a cell according to any one of claims 18 to 21, a cell prepared by the method according to any one of claims 22 to 26, or a chimeric molecule according to claim 28 for use in treating and / or preventing a disease associated with expression of WT1, wherein optionally, the disease associated with expression of WT1 is a proliferative disease, preferably, the proliferative disease is a hematological malignancy or a solid tumor, preferably, the hematological malignancy is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or a combination thereof. or preferably, the solid tumor is selected from the group consisting of lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, synovial cancer, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial carcinoma, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

31. A method for treating and / or preventing a disease associated with expression of WT1, the method comprising the step of administering to a subject in need thereof a TCR according to any one of claims 1 to 11, an isolated polynucleotide according to any one of claims 13 to 15, a vector according to claim 16 or 17, a cell according to any one of claims 18 to 21, a cell prepared by the method according to any one of claims 22 to 26, or a chimeric molecule according to claim 28.

32. The method of claim 31, wherein the disease associated with expression of WT1 is a proliferative disease, preferably the proliferative disease is a hematological malignancy or a solid tumor, preferably the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma; or preferably the solid tumor is selected from the group consisting of lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial carcinoma, biliary tract cancer, glioblastoma, cervical cancer, mesothelioma, and colorectal cancer.

33. 1. An isolated immunogenic WT1 peptide comprising an amino acid sequence selected from the group consisting of EPASQHTLRSG (SEQ ID NO:123), YESDNHTTPIL (SEQ ID NO:126), NHTTPILCGAQYRIH (SEQ ID NO:127), QCLSAFTVHFSGQFT (SEQ ID NO:118), EDPMGQQGSLGEQQY (SEQ ID NO:119), SQLECMTWNQMNLGA (SEQ ID NO:120), APVLDFAPPGA (SEQ ID NO:117), NQMNLGATLKG (SEQ ID NO:250), DPGGIWAKLGAAEAS (SEQ ID NO:251), NHTTPILCGAQYRIH (SEQ ID NO:252), KRHQRRHTGVKPFQC (SEQ ID NO:253), PSCQKKFARSDELVR (SEQ ID NO:254), and each of these variants having up to three amino acid substitutions, additions, or deletions.

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